DMD35956
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The intestine as an important contributor to prasugrel active metabolite formation
in vivo
Katsunobu Hagihara Miho Kazui Hidenori Ikenaga Toshihiko Nanba Kiichi
Fusegawa Takashi Izumi Toshihiko Ikeda and Atsushi Kurihara
Drug Metabolism amp Pharmacokinetics Research Laboratories (KH MK TI and
AK) and Pharmacology Research Laboratories (HI TN and KF) Daiichi Sankyo
Co Ltd Tokyo Japan Yokohama College of Pharmacy Yokohama Japan (TI)
DMD Fast Forward Published on December 28 2010 as doi101124dmd110035956
Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics
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Running Title Prasugrel active metabolite formation in intestine
Address correspondance to
Katsunobu Hagihara
Drug Metabolism amp Pharmacokinetics Research Laboratories Daiichi Sankyo Co Ltd
1-2-58 Hiromachi Shinagawa-Ku Tokyo 140-8710 Japan
Tel +81-3-3492-3131
Fax +81-3-5436-8567
E-mail hagiharakatsunobufcdaiichisankyocojp
Text Pages 14
Tables 3
Figures 6
References 21
Abstract 168 words
Introduction 334 words
Discussion 807 words
Abbreviations prasugrel
2-acetoxy-5-(α-cyclopropylcarbonyl-2-fluorobenzyl)-4567-tetrahydrothieno[32-c]pyr
idine S9 9000g supernatant
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ABSTRACT
Prasugrel a thienopyridine antiplatelet agent undergoes rapid hydrolysis in vivo to a
thiolactone intermediate R-95913 which is further converted to a pharmacologically
active metabolite R-138727 by oxidation via cytochromes P450 In this study we
investigated how much the intestine and liver contribute to the formation of R-95913 and
R-138727 after intraduodenal administration of prasugrel (1 mgkg) to portal vein- and
hepatic vein-cannulated dogs The areas under the plasma concentration-time curve up
to 2 h (AUC0-2h) of R-95913 in the portal hepatic and systemic veins were 525 32 and 17
nghmL respectively and those of R-138727 were 564 529 and 495 nghmL
respectively The dosed prasugrel was absorbed and then converted to R-95913 and
R-138727 by 93 and 13 respectively in the intestine In the liver 23 of the
R-95913 which passed through the intestine was converted to R-138727 In conclusion
this is the first report to directly demonstrate that the conversion of prasugrel to R-138727
in the intestine is comparable to that converted in the liver of dogs
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INTRODUCTION
Prasugrel (Effientreg in the US and Efientreg in EU) clopidogrel (PlavixregIscoverreg) and
ticlopidine (TiclidTM) are thienopyridine antiplatelet agents Prasugrel is indicated to
reduce the rate of thrombotic cardiovascular events and stent thrombosis in patients
with acute coronary syndrome that are undergoing percutaneous coronary intervention
(Wiviott et al 2007 Effient package insert) The thienopyridines are prodrugs that
are converted in vivo to their pharmacologically active metabolites possessing a thiol
group via a corresponding thiolactone metabolite (Farid et al 2010) In clinical trials
prasugrel achieved greater and faster antiplatelet effect than clopidogrel (Payne et al
2007 Wallentin et al 2008) Such responses to prasugrel are attributed to higher and
faster exposure to its active metabolite R-138727 than clopidogrelrsquos (Sugidachi et al
2007 Ernest et al 2008) Prasugrel is rapidly hydrolyzed to a thiolactone
intermediate R-95913 mainly by human carboxylesterase 2 during absorption through
the gastrointestinal tract (Williams et al 2008) R-95913 is metabolized to R-138727
by cytochrome P450 (CYP) isoforms and the main contributors are CYP3A4 and
CYP2B6 (38-70 and 2-36 respectively) with smaller contributions by CYP2C9
and CYP2C19 (14-19 and 8-11 respectively Rehmel et al 2006) CYP3A5 is as
effective as CYP3A4 in converting R-95913 to R-138727 (Baker et al 2008)
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indicating that CYP3A is a key isoform for R-138727 formation Considering that
CYP3A represents about 80 of the intestinal CYP forms (Paine et al 2006) a large
proportion of R-138727 could be formed during first-pass metabolism in the intestine
We previously detected R-138727 in the portal vein after intraduodenal administration
of prasugrel to rats (Hagihara et al 2009) possibly indicating the formation of
R-138727 in the intestine However that does not prove unequivocally the intestinal
contribution because the appearance of R-138727 in the portal vein was slow and could
have been derived from the circulation after formation in the liver (Hagihara et al
2009)
In this study we determined quantitatively the contribution ratio of the intestine and
liver to the formation of the intermediate R-95913 and the active metabolite R-138727
from prasugrel in dogs
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Materials and Methods
Materials Prasugrel prasugrel thiolactone (R-95913) and prasugrel active metabolite
(R-138727) shown in Figure 1 were synthesized by Ube Industries Ltd (Ube Japan)
Phenacetine and NN-dimethylacetamide were purchased from Sigma-Aldrich (St
Louis MO) Polyethylene glycol 400 was obtained from Wako Pure Chemical
Industries Ltd (Osaka Japan) Methoxyphenacyl bromide was obtained from Tokyo
Kasei Kogyo Co Ltd (Tokyo Japan) Human intestinal microsomes dog intestinal
microsomes and dog liver microsomes were purchased from Tissue Transformation
Technologies Inc (Edison NJ) Biopredic International (Rennes France) and
XenoTech (Lenexa KS) respectively All other chemicals and reagents were
commercially available and of the highest grade
Preparation of dosing formulations Prasugrel was dissolved in 5
NN-dimethylacetamide95 polyethylene glycol 400 (vv) solution at concentrations
of 3 mgmL Solution was prepared immediately before use
Experimental animals Male Beagle dogs (n=6) at ages of 2-3 years were obtained
from Nosan Corporation and were acclimatized in a controlled animal area set at
23plusmn2degC (acceptable range 20-26degC) and relative humidity of 55plusmn5 (acceptable range
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30-70) under a 12-h cycle of lightdark artificial lighting (lighting time 700 AM -
700 PM) Food (DS-A Oriental Yeast Co Ltd Tokyo Japan) and water were
provided freely Dogs weighing 9 to 12 kg that underwent the portal- and
hepatic-vein cannulating surgery were used in the experiments and were fasted for
about 19 h prior to prasugrel administration All animal experiments were carried out
according to the guidelines provided by the Institutional Animal Care and Use
Committee of Daiichi Sankyo Co Ltd
Portal- and hepatic-vein cannulated dogs Under pentobarbital anesthesia the
abdomen was incised on the median line The portal vein was clamped onto the
liver-leaning and intestine-leaning sites of the cannulation point into which the catheter
(Medicut LCV-UK kit single lumen outer diameter 16G length 70 cm Sherwood
Medical Company St Louis MO) was inserted A purse string ligature was applied
and the clamps were released After the portal vein cannulation the same catheter was
also inserted into the hepatic vein and a purse string ligature was applied The
catheters were conjugated with each injection port which was embedded
subcutaneously
Animal experiments From the jugular vein 2 mL of blood was extracted before
dosing The dosing solution of prasugrel was intraduodenally administered to the
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cannulated dogs (n=6) at a dose level of 1 mgkg After about 1 mL of blood drainage
from the portal and hepatic veins via the injection port the syringe was changed to
another one and 1 mL of blood was collected with the heparinized syringe at 1 5 10
15 30 60 and 120 min post-dose The blood in the cannula was pushed back by the
injection of 2 mL of heparin solution in saline (100 unitsmL) At each time point 1
mL of blood was also collected from the jugular vein The blood samples were
immediately centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd Tokyo
Japan) to collect plasma samples A total of 50 μL of plasma was mixed with 100 μL
of acetonitrile and 50 μL of the IS solution (4 μM phenacetin in acetonitrile) and the
mixture was centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd) A
total of 10 μL of the supernatant was injected to LC-MS
Quantitation of R-95913 and R-138727 The quantitation of R-95913 and R-138727
in dog plasma was carried out on an Alliance HPLC system consisting of a 2690
Separations Module (Waters Co Milford MA) coupled to a Quattro LC MS system
(Micromass Ltd Milford MA) with the ESI source in positive ion mode The mobile
phase containing acetonitrile 5 mM ammonium acetate and formic acid (326802
vvv) was applied onto the column A total of 10 μL of each sample was injected onto
a CAPCEL PAK C18 column (5 μm 150 mm times 15 mm Shiseido Co Ltd Tokyo
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Japan) The operating parameters of the MS detector were set as follows capillary
voltage 35 kV ion source temperature 120degC and desolvation temperature 350degC
The detection was performed in the multiple reaction monitoring (MRM) mode The
concentrations of each analyte in the samples were calculated using the computer
software MassLynx (Version 34 Micromass Ltd)
Pharmacokinetic analysis The pharmacokinetic parameters were calculated using the
computer program WinNonlin Professional (ver 401 Pharsight Corp Mountain View
CA) based on the non-compartmental method The area under the plasma
concentration-time curve up to 2 h (AUC0-2h) was calculated by the trapezoidal method
and are expressed as the meanplusmnSD
Calculation of availabililty of R-95913 The availability of R-95913 was calculated
using the following equations
Fa Fg prasrarr913 = Qp Rb (AUCpor913 - AUCsys913) Dose (1)
Fh 913 = AUChep913 AUCpor913 (2)
where Fa Fg prasrarr913 and Fh 913 are fraction of intestinal absorption of prasugrel fraction
of prasugrel hydrolysis to R-95913 in the intestine and availability of R-95913 in the
liver respectively AUCpor913 AUChep913 and AUCsys913 indicate the AUC0-2h values
of R-95913 in the portal hepatic and systemic veins respectively Qp is portal blood
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flow and Rb is the bloodplasma concentration ratio Fa Qp and Rb are set as 0968
(Hagihara et al 2007) 521 mLmin (Hoshino et al 1986) and 0597 (In-house data
Daiichi Sankyo Co Ltd) respectively
Calculation of availabililty of R-138727 The availability of R-138727 was calculated
using the following equations
Fa Fg prasrarr727 = Qp Rb (AUCpor727 - AUCsys727) Dose (3)
Fm 913rarr727 = (AUChep727 - AUCpor727 Fh 727 ) AUCpor913 (4)
Fa Fg prasrarr727 Fh 727 + Fa Fg prasrarr913 Fm 913rarr727 = Relative BA (5)
where Fg prasrarr727 Fm 913rarr727 and Fh 727 are fraction of prasugrel conversion to R-138727
in the intestine fraction of R-95913 conversion to R-138727 in the liver and availability
of R-138727 in the liver respectively AUCpor727 AUChep727 and AUCsys727 indicate
the AUC0-2h values of R-138727 in the portal hepatic and systemic veins respectively
Relative BA means the relative bioavailability of R-138727 after oral dosing of
prasugrel to dogs and was set as 248 (Hagihara et al 2009)
Formation of R-95913 in dog and human intestinal S9 The mixture (total volume
2475 μL) in triplicate contained potassium phosphate buffer (7 mM pH 74) an
NADPH generating system containing 25 mM β-NADP 25 mM G-6-P 05 unitsmL
G-6-PDH and 10 mM MgCl2 5 mM glutathione and dog or human intestinal S9 (10 mg
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proteinmL each) The mixture was preincubated at 37degC for 5 min and 25 μL of
prasugrel (final concentration 250 μM) was added to the mixture which was incubated
at 37degC for 0 5 15 30 and 60 min Ninety μL of ethanol was added to 30 μL of the
reaction mixture to stop the reaction at each time point and the mixture was centrifuged
(15000g 3 min 4degC) Twenty five μL of the supernatant was injected into the HPLC
system HPLC was performed using YMC-ODS A-302 (46 mm id times150 mm)
pumped at flow rate of 05 mLmin with a mobile phase consisting of acetonitrile
isopropyl alcohol distilled water trifluoroacetic acid (51283001 vvvv)
Absorption of the column effluent was monitored at 220 nm to detect the metabolites
Lower limit of quantification for R-95913 was 1 μM
Formation of R-138727 in human and dog intestinal and dog liver microsomes The
mixture (total volume 110 μL) in triplicate contained potassium phosphate buffer (43
mM pH 74) an NADPH generating system (13 mM NADP+ 33 mM
glucose-6-phosphate 04 unitsmL glucose-6-phosphate dehydrogenase and 33 mM
MgCl2) 5 mM glutathione and human or dog intestinal or dog liver microsomes (1 mg
proteinmL each) The mixture was preincubated at 37degC for 5 min and 11 μL of
R-95913 (final concentrations ranging from 16 to 200 μM) was added to the mixture
After incubation at 37degC for 15 min 200 μL of 5 mM methoxyphenacyl bromide
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solution and 100 μL of the IS solution (100 ngmL R-135766) in acetonitrile were added
to 100 μL of the reaction mixture to stop the reaction and the mixture was left for 10
min at room temperature to derivaitze a thiol moiety of R-138727 The mixture was
extracted by a solid phase extract column (Captiva Varian Inc Palo Alto CA) The
assay of R-138727 was performed following the methods previously reported (Hagihara
et al 2009) Separation of the analytes by HPLC was conducted using an
Alliance2690 Separations Module (Waters Co Milford MA) Mass spectra were
determined using a Quattro LC MSMS system (Micromass Ltd Milford MA) in the
positive ion detection mode using an ESI-interface A lower limit of quantification
was set at 16 nM Data acquisition and analyses were performed using MassLynx
software (Version 40 Micromass Ltd)
Data handling The formation pattern of R-138727 in human or dog intestinal or dog
liver microsomes appeared monophasic in each Eadie-Hofstee plot (data not shown)
Therefore the data were fitted to eq 1 using WinNonlin Professional (version 401
Pharsight Corp)
V = Vmax times S (Km + S) (6)
where S Km and Vmax is the substrate concentration Michaelis-Menten constant and
maximal formation rate respectively The intrinsic clearance (CLint) was calculated as
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a ratio of Vmax to Km These parameters are expressed as meanplusmnSD
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Results
Plasma concentrations and AUC0-2hs after intraduodenal administration of
prasugrel to cannulated dogs The plasma concentrations of R-95913 and R-138727
were determined by LC-MS after intraduodenal administration of prasugrel to the portal
and hepatic vein cannulated dogs at a dose of 1 mgkg Plasma concentrations of
prasugrel were not analyzed as prasugrel was not detected unmodified even in the portal
vein in a previous study (Hagihara et al 2009) The plasma concentration-time
profiles of R-95913 and R-138727 are shown in Figures 2 and 3 respectively The
AUC0-2h value of R-95913 in the portal vein (525plusmn234 nghmL) was much higher than
those in hepatic and systemic veins (32plusmn17 and 17plusmn8 nghmL respectively) The
AUC0-2h values of R-138727 in the portal hepatic and systemic veins were 564plusmn187
nghmL 529plusmn136 nghmL and 495plusmn233 nghmL respectively The bimodal peaks
were detected in the plasma concentration-time profiles of R-95913 (Figure 2) This
may indicate reabsorption of R-95913 from the intestine as in the case of rats which
showed enterohepatic circulation of 14C-prasugrel (Hagihara et al 2007)
Availability of R-95913 and R-138727 in the intestine and the liver The FaFg prasrarr
913 and Fh 913 values were 080plusmn023 and 008plusmn006 respectively (Table 1) The
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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Running Title Prasugrel active metabolite formation in intestine
Address correspondance to
Katsunobu Hagihara
Drug Metabolism amp Pharmacokinetics Research Laboratories Daiichi Sankyo Co Ltd
1-2-58 Hiromachi Shinagawa-Ku Tokyo 140-8710 Japan
Tel +81-3-3492-3131
Fax +81-3-5436-8567
E-mail hagiharakatsunobufcdaiichisankyocojp
Text Pages 14
Tables 3
Figures 6
References 21
Abstract 168 words
Introduction 334 words
Discussion 807 words
Abbreviations prasugrel
2-acetoxy-5-(α-cyclopropylcarbonyl-2-fluorobenzyl)-4567-tetrahydrothieno[32-c]pyr
idine S9 9000g supernatant
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ABSTRACT
Prasugrel a thienopyridine antiplatelet agent undergoes rapid hydrolysis in vivo to a
thiolactone intermediate R-95913 which is further converted to a pharmacologically
active metabolite R-138727 by oxidation via cytochromes P450 In this study we
investigated how much the intestine and liver contribute to the formation of R-95913 and
R-138727 after intraduodenal administration of prasugrel (1 mgkg) to portal vein- and
hepatic vein-cannulated dogs The areas under the plasma concentration-time curve up
to 2 h (AUC0-2h) of R-95913 in the portal hepatic and systemic veins were 525 32 and 17
nghmL respectively and those of R-138727 were 564 529 and 495 nghmL
respectively The dosed prasugrel was absorbed and then converted to R-95913 and
R-138727 by 93 and 13 respectively in the intestine In the liver 23 of the
R-95913 which passed through the intestine was converted to R-138727 In conclusion
this is the first report to directly demonstrate that the conversion of prasugrel to R-138727
in the intestine is comparable to that converted in the liver of dogs
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INTRODUCTION
Prasugrel (Effientreg in the US and Efientreg in EU) clopidogrel (PlavixregIscoverreg) and
ticlopidine (TiclidTM) are thienopyridine antiplatelet agents Prasugrel is indicated to
reduce the rate of thrombotic cardiovascular events and stent thrombosis in patients
with acute coronary syndrome that are undergoing percutaneous coronary intervention
(Wiviott et al 2007 Effient package insert) The thienopyridines are prodrugs that
are converted in vivo to their pharmacologically active metabolites possessing a thiol
group via a corresponding thiolactone metabolite (Farid et al 2010) In clinical trials
prasugrel achieved greater and faster antiplatelet effect than clopidogrel (Payne et al
2007 Wallentin et al 2008) Such responses to prasugrel are attributed to higher and
faster exposure to its active metabolite R-138727 than clopidogrelrsquos (Sugidachi et al
2007 Ernest et al 2008) Prasugrel is rapidly hydrolyzed to a thiolactone
intermediate R-95913 mainly by human carboxylesterase 2 during absorption through
the gastrointestinal tract (Williams et al 2008) R-95913 is metabolized to R-138727
by cytochrome P450 (CYP) isoforms and the main contributors are CYP3A4 and
CYP2B6 (38-70 and 2-36 respectively) with smaller contributions by CYP2C9
and CYP2C19 (14-19 and 8-11 respectively Rehmel et al 2006) CYP3A5 is as
effective as CYP3A4 in converting R-95913 to R-138727 (Baker et al 2008)
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indicating that CYP3A is a key isoform for R-138727 formation Considering that
CYP3A represents about 80 of the intestinal CYP forms (Paine et al 2006) a large
proportion of R-138727 could be formed during first-pass metabolism in the intestine
We previously detected R-138727 in the portal vein after intraduodenal administration
of prasugrel to rats (Hagihara et al 2009) possibly indicating the formation of
R-138727 in the intestine However that does not prove unequivocally the intestinal
contribution because the appearance of R-138727 in the portal vein was slow and could
have been derived from the circulation after formation in the liver (Hagihara et al
2009)
In this study we determined quantitatively the contribution ratio of the intestine and
liver to the formation of the intermediate R-95913 and the active metabolite R-138727
from prasugrel in dogs
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Materials and Methods
Materials Prasugrel prasugrel thiolactone (R-95913) and prasugrel active metabolite
(R-138727) shown in Figure 1 were synthesized by Ube Industries Ltd (Ube Japan)
Phenacetine and NN-dimethylacetamide were purchased from Sigma-Aldrich (St
Louis MO) Polyethylene glycol 400 was obtained from Wako Pure Chemical
Industries Ltd (Osaka Japan) Methoxyphenacyl bromide was obtained from Tokyo
Kasei Kogyo Co Ltd (Tokyo Japan) Human intestinal microsomes dog intestinal
microsomes and dog liver microsomes were purchased from Tissue Transformation
Technologies Inc (Edison NJ) Biopredic International (Rennes France) and
XenoTech (Lenexa KS) respectively All other chemicals and reagents were
commercially available and of the highest grade
Preparation of dosing formulations Prasugrel was dissolved in 5
NN-dimethylacetamide95 polyethylene glycol 400 (vv) solution at concentrations
of 3 mgmL Solution was prepared immediately before use
Experimental animals Male Beagle dogs (n=6) at ages of 2-3 years were obtained
from Nosan Corporation and were acclimatized in a controlled animal area set at
23plusmn2degC (acceptable range 20-26degC) and relative humidity of 55plusmn5 (acceptable range
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30-70) under a 12-h cycle of lightdark artificial lighting (lighting time 700 AM -
700 PM) Food (DS-A Oriental Yeast Co Ltd Tokyo Japan) and water were
provided freely Dogs weighing 9 to 12 kg that underwent the portal- and
hepatic-vein cannulating surgery were used in the experiments and were fasted for
about 19 h prior to prasugrel administration All animal experiments were carried out
according to the guidelines provided by the Institutional Animal Care and Use
Committee of Daiichi Sankyo Co Ltd
Portal- and hepatic-vein cannulated dogs Under pentobarbital anesthesia the
abdomen was incised on the median line The portal vein was clamped onto the
liver-leaning and intestine-leaning sites of the cannulation point into which the catheter
(Medicut LCV-UK kit single lumen outer diameter 16G length 70 cm Sherwood
Medical Company St Louis MO) was inserted A purse string ligature was applied
and the clamps were released After the portal vein cannulation the same catheter was
also inserted into the hepatic vein and a purse string ligature was applied The
catheters were conjugated with each injection port which was embedded
subcutaneously
Animal experiments From the jugular vein 2 mL of blood was extracted before
dosing The dosing solution of prasugrel was intraduodenally administered to the
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cannulated dogs (n=6) at a dose level of 1 mgkg After about 1 mL of blood drainage
from the portal and hepatic veins via the injection port the syringe was changed to
another one and 1 mL of blood was collected with the heparinized syringe at 1 5 10
15 30 60 and 120 min post-dose The blood in the cannula was pushed back by the
injection of 2 mL of heparin solution in saline (100 unitsmL) At each time point 1
mL of blood was also collected from the jugular vein The blood samples were
immediately centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd Tokyo
Japan) to collect plasma samples A total of 50 μL of plasma was mixed with 100 μL
of acetonitrile and 50 μL of the IS solution (4 μM phenacetin in acetonitrile) and the
mixture was centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd) A
total of 10 μL of the supernatant was injected to LC-MS
Quantitation of R-95913 and R-138727 The quantitation of R-95913 and R-138727
in dog plasma was carried out on an Alliance HPLC system consisting of a 2690
Separations Module (Waters Co Milford MA) coupled to a Quattro LC MS system
(Micromass Ltd Milford MA) with the ESI source in positive ion mode The mobile
phase containing acetonitrile 5 mM ammonium acetate and formic acid (326802
vvv) was applied onto the column A total of 10 μL of each sample was injected onto
a CAPCEL PAK C18 column (5 μm 150 mm times 15 mm Shiseido Co Ltd Tokyo
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Japan) The operating parameters of the MS detector were set as follows capillary
voltage 35 kV ion source temperature 120degC and desolvation temperature 350degC
The detection was performed in the multiple reaction monitoring (MRM) mode The
concentrations of each analyte in the samples were calculated using the computer
software MassLynx (Version 34 Micromass Ltd)
Pharmacokinetic analysis The pharmacokinetic parameters were calculated using the
computer program WinNonlin Professional (ver 401 Pharsight Corp Mountain View
CA) based on the non-compartmental method The area under the plasma
concentration-time curve up to 2 h (AUC0-2h) was calculated by the trapezoidal method
and are expressed as the meanplusmnSD
Calculation of availabililty of R-95913 The availability of R-95913 was calculated
using the following equations
Fa Fg prasrarr913 = Qp Rb (AUCpor913 - AUCsys913) Dose (1)
Fh 913 = AUChep913 AUCpor913 (2)
where Fa Fg prasrarr913 and Fh 913 are fraction of intestinal absorption of prasugrel fraction
of prasugrel hydrolysis to R-95913 in the intestine and availability of R-95913 in the
liver respectively AUCpor913 AUChep913 and AUCsys913 indicate the AUC0-2h values
of R-95913 in the portal hepatic and systemic veins respectively Qp is portal blood
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flow and Rb is the bloodplasma concentration ratio Fa Qp and Rb are set as 0968
(Hagihara et al 2007) 521 mLmin (Hoshino et al 1986) and 0597 (In-house data
Daiichi Sankyo Co Ltd) respectively
Calculation of availabililty of R-138727 The availability of R-138727 was calculated
using the following equations
Fa Fg prasrarr727 = Qp Rb (AUCpor727 - AUCsys727) Dose (3)
Fm 913rarr727 = (AUChep727 - AUCpor727 Fh 727 ) AUCpor913 (4)
Fa Fg prasrarr727 Fh 727 + Fa Fg prasrarr913 Fm 913rarr727 = Relative BA (5)
where Fg prasrarr727 Fm 913rarr727 and Fh 727 are fraction of prasugrel conversion to R-138727
in the intestine fraction of R-95913 conversion to R-138727 in the liver and availability
of R-138727 in the liver respectively AUCpor727 AUChep727 and AUCsys727 indicate
the AUC0-2h values of R-138727 in the portal hepatic and systemic veins respectively
Relative BA means the relative bioavailability of R-138727 after oral dosing of
prasugrel to dogs and was set as 248 (Hagihara et al 2009)
Formation of R-95913 in dog and human intestinal S9 The mixture (total volume
2475 μL) in triplicate contained potassium phosphate buffer (7 mM pH 74) an
NADPH generating system containing 25 mM β-NADP 25 mM G-6-P 05 unitsmL
G-6-PDH and 10 mM MgCl2 5 mM glutathione and dog or human intestinal S9 (10 mg
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proteinmL each) The mixture was preincubated at 37degC for 5 min and 25 μL of
prasugrel (final concentration 250 μM) was added to the mixture which was incubated
at 37degC for 0 5 15 30 and 60 min Ninety μL of ethanol was added to 30 μL of the
reaction mixture to stop the reaction at each time point and the mixture was centrifuged
(15000g 3 min 4degC) Twenty five μL of the supernatant was injected into the HPLC
system HPLC was performed using YMC-ODS A-302 (46 mm id times150 mm)
pumped at flow rate of 05 mLmin with a mobile phase consisting of acetonitrile
isopropyl alcohol distilled water trifluoroacetic acid (51283001 vvvv)
Absorption of the column effluent was monitored at 220 nm to detect the metabolites
Lower limit of quantification for R-95913 was 1 μM
Formation of R-138727 in human and dog intestinal and dog liver microsomes The
mixture (total volume 110 μL) in triplicate contained potassium phosphate buffer (43
mM pH 74) an NADPH generating system (13 mM NADP+ 33 mM
glucose-6-phosphate 04 unitsmL glucose-6-phosphate dehydrogenase and 33 mM
MgCl2) 5 mM glutathione and human or dog intestinal or dog liver microsomes (1 mg
proteinmL each) The mixture was preincubated at 37degC for 5 min and 11 μL of
R-95913 (final concentrations ranging from 16 to 200 μM) was added to the mixture
After incubation at 37degC for 15 min 200 μL of 5 mM methoxyphenacyl bromide
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solution and 100 μL of the IS solution (100 ngmL R-135766) in acetonitrile were added
to 100 μL of the reaction mixture to stop the reaction and the mixture was left for 10
min at room temperature to derivaitze a thiol moiety of R-138727 The mixture was
extracted by a solid phase extract column (Captiva Varian Inc Palo Alto CA) The
assay of R-138727 was performed following the methods previously reported (Hagihara
et al 2009) Separation of the analytes by HPLC was conducted using an
Alliance2690 Separations Module (Waters Co Milford MA) Mass spectra were
determined using a Quattro LC MSMS system (Micromass Ltd Milford MA) in the
positive ion detection mode using an ESI-interface A lower limit of quantification
was set at 16 nM Data acquisition and analyses were performed using MassLynx
software (Version 40 Micromass Ltd)
Data handling The formation pattern of R-138727 in human or dog intestinal or dog
liver microsomes appeared monophasic in each Eadie-Hofstee plot (data not shown)
Therefore the data were fitted to eq 1 using WinNonlin Professional (version 401
Pharsight Corp)
V = Vmax times S (Km + S) (6)
where S Km and Vmax is the substrate concentration Michaelis-Menten constant and
maximal formation rate respectively The intrinsic clearance (CLint) was calculated as
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a ratio of Vmax to Km These parameters are expressed as meanplusmnSD
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Results
Plasma concentrations and AUC0-2hs after intraduodenal administration of
prasugrel to cannulated dogs The plasma concentrations of R-95913 and R-138727
were determined by LC-MS after intraduodenal administration of prasugrel to the portal
and hepatic vein cannulated dogs at a dose of 1 mgkg Plasma concentrations of
prasugrel were not analyzed as prasugrel was not detected unmodified even in the portal
vein in a previous study (Hagihara et al 2009) The plasma concentration-time
profiles of R-95913 and R-138727 are shown in Figures 2 and 3 respectively The
AUC0-2h value of R-95913 in the portal vein (525plusmn234 nghmL) was much higher than
those in hepatic and systemic veins (32plusmn17 and 17plusmn8 nghmL respectively) The
AUC0-2h values of R-138727 in the portal hepatic and systemic veins were 564plusmn187
nghmL 529plusmn136 nghmL and 495plusmn233 nghmL respectively The bimodal peaks
were detected in the plasma concentration-time profiles of R-95913 (Figure 2) This
may indicate reabsorption of R-95913 from the intestine as in the case of rats which
showed enterohepatic circulation of 14C-prasugrel (Hagihara et al 2007)
Availability of R-95913 and R-138727 in the intestine and the liver The FaFg prasrarr
913 and Fh 913 values were 080plusmn023 and 008plusmn006 respectively (Table 1) The
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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ABSTRACT
Prasugrel a thienopyridine antiplatelet agent undergoes rapid hydrolysis in vivo to a
thiolactone intermediate R-95913 which is further converted to a pharmacologically
active metabolite R-138727 by oxidation via cytochromes P450 In this study we
investigated how much the intestine and liver contribute to the formation of R-95913 and
R-138727 after intraduodenal administration of prasugrel (1 mgkg) to portal vein- and
hepatic vein-cannulated dogs The areas under the plasma concentration-time curve up
to 2 h (AUC0-2h) of R-95913 in the portal hepatic and systemic veins were 525 32 and 17
nghmL respectively and those of R-138727 were 564 529 and 495 nghmL
respectively The dosed prasugrel was absorbed and then converted to R-95913 and
R-138727 by 93 and 13 respectively in the intestine In the liver 23 of the
R-95913 which passed through the intestine was converted to R-138727 In conclusion
this is the first report to directly demonstrate that the conversion of prasugrel to R-138727
in the intestine is comparable to that converted in the liver of dogs
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INTRODUCTION
Prasugrel (Effientreg in the US and Efientreg in EU) clopidogrel (PlavixregIscoverreg) and
ticlopidine (TiclidTM) are thienopyridine antiplatelet agents Prasugrel is indicated to
reduce the rate of thrombotic cardiovascular events and stent thrombosis in patients
with acute coronary syndrome that are undergoing percutaneous coronary intervention
(Wiviott et al 2007 Effient package insert) The thienopyridines are prodrugs that
are converted in vivo to their pharmacologically active metabolites possessing a thiol
group via a corresponding thiolactone metabolite (Farid et al 2010) In clinical trials
prasugrel achieved greater and faster antiplatelet effect than clopidogrel (Payne et al
2007 Wallentin et al 2008) Such responses to prasugrel are attributed to higher and
faster exposure to its active metabolite R-138727 than clopidogrelrsquos (Sugidachi et al
2007 Ernest et al 2008) Prasugrel is rapidly hydrolyzed to a thiolactone
intermediate R-95913 mainly by human carboxylesterase 2 during absorption through
the gastrointestinal tract (Williams et al 2008) R-95913 is metabolized to R-138727
by cytochrome P450 (CYP) isoforms and the main contributors are CYP3A4 and
CYP2B6 (38-70 and 2-36 respectively) with smaller contributions by CYP2C9
and CYP2C19 (14-19 and 8-11 respectively Rehmel et al 2006) CYP3A5 is as
effective as CYP3A4 in converting R-95913 to R-138727 (Baker et al 2008)
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indicating that CYP3A is a key isoform for R-138727 formation Considering that
CYP3A represents about 80 of the intestinal CYP forms (Paine et al 2006) a large
proportion of R-138727 could be formed during first-pass metabolism in the intestine
We previously detected R-138727 in the portal vein after intraduodenal administration
of prasugrel to rats (Hagihara et al 2009) possibly indicating the formation of
R-138727 in the intestine However that does not prove unequivocally the intestinal
contribution because the appearance of R-138727 in the portal vein was slow and could
have been derived from the circulation after formation in the liver (Hagihara et al
2009)
In this study we determined quantitatively the contribution ratio of the intestine and
liver to the formation of the intermediate R-95913 and the active metabolite R-138727
from prasugrel in dogs
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Materials and Methods
Materials Prasugrel prasugrel thiolactone (R-95913) and prasugrel active metabolite
(R-138727) shown in Figure 1 were synthesized by Ube Industries Ltd (Ube Japan)
Phenacetine and NN-dimethylacetamide were purchased from Sigma-Aldrich (St
Louis MO) Polyethylene glycol 400 was obtained from Wako Pure Chemical
Industries Ltd (Osaka Japan) Methoxyphenacyl bromide was obtained from Tokyo
Kasei Kogyo Co Ltd (Tokyo Japan) Human intestinal microsomes dog intestinal
microsomes and dog liver microsomes were purchased from Tissue Transformation
Technologies Inc (Edison NJ) Biopredic International (Rennes France) and
XenoTech (Lenexa KS) respectively All other chemicals and reagents were
commercially available and of the highest grade
Preparation of dosing formulations Prasugrel was dissolved in 5
NN-dimethylacetamide95 polyethylene glycol 400 (vv) solution at concentrations
of 3 mgmL Solution was prepared immediately before use
Experimental animals Male Beagle dogs (n=6) at ages of 2-3 years were obtained
from Nosan Corporation and were acclimatized in a controlled animal area set at
23plusmn2degC (acceptable range 20-26degC) and relative humidity of 55plusmn5 (acceptable range
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30-70) under a 12-h cycle of lightdark artificial lighting (lighting time 700 AM -
700 PM) Food (DS-A Oriental Yeast Co Ltd Tokyo Japan) and water were
provided freely Dogs weighing 9 to 12 kg that underwent the portal- and
hepatic-vein cannulating surgery were used in the experiments and were fasted for
about 19 h prior to prasugrel administration All animal experiments were carried out
according to the guidelines provided by the Institutional Animal Care and Use
Committee of Daiichi Sankyo Co Ltd
Portal- and hepatic-vein cannulated dogs Under pentobarbital anesthesia the
abdomen was incised on the median line The portal vein was clamped onto the
liver-leaning and intestine-leaning sites of the cannulation point into which the catheter
(Medicut LCV-UK kit single lumen outer diameter 16G length 70 cm Sherwood
Medical Company St Louis MO) was inserted A purse string ligature was applied
and the clamps were released After the portal vein cannulation the same catheter was
also inserted into the hepatic vein and a purse string ligature was applied The
catheters were conjugated with each injection port which was embedded
subcutaneously
Animal experiments From the jugular vein 2 mL of blood was extracted before
dosing The dosing solution of prasugrel was intraduodenally administered to the
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cannulated dogs (n=6) at a dose level of 1 mgkg After about 1 mL of blood drainage
from the portal and hepatic veins via the injection port the syringe was changed to
another one and 1 mL of blood was collected with the heparinized syringe at 1 5 10
15 30 60 and 120 min post-dose The blood in the cannula was pushed back by the
injection of 2 mL of heparin solution in saline (100 unitsmL) At each time point 1
mL of blood was also collected from the jugular vein The blood samples were
immediately centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd Tokyo
Japan) to collect plasma samples A total of 50 μL of plasma was mixed with 100 μL
of acetonitrile and 50 μL of the IS solution (4 μM phenacetin in acetonitrile) and the
mixture was centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd) A
total of 10 μL of the supernatant was injected to LC-MS
Quantitation of R-95913 and R-138727 The quantitation of R-95913 and R-138727
in dog plasma was carried out on an Alliance HPLC system consisting of a 2690
Separations Module (Waters Co Milford MA) coupled to a Quattro LC MS system
(Micromass Ltd Milford MA) with the ESI source in positive ion mode The mobile
phase containing acetonitrile 5 mM ammonium acetate and formic acid (326802
vvv) was applied onto the column A total of 10 μL of each sample was injected onto
a CAPCEL PAK C18 column (5 μm 150 mm times 15 mm Shiseido Co Ltd Tokyo
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Japan) The operating parameters of the MS detector were set as follows capillary
voltage 35 kV ion source temperature 120degC and desolvation temperature 350degC
The detection was performed in the multiple reaction monitoring (MRM) mode The
concentrations of each analyte in the samples were calculated using the computer
software MassLynx (Version 34 Micromass Ltd)
Pharmacokinetic analysis The pharmacokinetic parameters were calculated using the
computer program WinNonlin Professional (ver 401 Pharsight Corp Mountain View
CA) based on the non-compartmental method The area under the plasma
concentration-time curve up to 2 h (AUC0-2h) was calculated by the trapezoidal method
and are expressed as the meanplusmnSD
Calculation of availabililty of R-95913 The availability of R-95913 was calculated
using the following equations
Fa Fg prasrarr913 = Qp Rb (AUCpor913 - AUCsys913) Dose (1)
Fh 913 = AUChep913 AUCpor913 (2)
where Fa Fg prasrarr913 and Fh 913 are fraction of intestinal absorption of prasugrel fraction
of prasugrel hydrolysis to R-95913 in the intestine and availability of R-95913 in the
liver respectively AUCpor913 AUChep913 and AUCsys913 indicate the AUC0-2h values
of R-95913 in the portal hepatic and systemic veins respectively Qp is portal blood
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flow and Rb is the bloodplasma concentration ratio Fa Qp and Rb are set as 0968
(Hagihara et al 2007) 521 mLmin (Hoshino et al 1986) and 0597 (In-house data
Daiichi Sankyo Co Ltd) respectively
Calculation of availabililty of R-138727 The availability of R-138727 was calculated
using the following equations
Fa Fg prasrarr727 = Qp Rb (AUCpor727 - AUCsys727) Dose (3)
Fm 913rarr727 = (AUChep727 - AUCpor727 Fh 727 ) AUCpor913 (4)
Fa Fg prasrarr727 Fh 727 + Fa Fg prasrarr913 Fm 913rarr727 = Relative BA (5)
where Fg prasrarr727 Fm 913rarr727 and Fh 727 are fraction of prasugrel conversion to R-138727
in the intestine fraction of R-95913 conversion to R-138727 in the liver and availability
of R-138727 in the liver respectively AUCpor727 AUChep727 and AUCsys727 indicate
the AUC0-2h values of R-138727 in the portal hepatic and systemic veins respectively
Relative BA means the relative bioavailability of R-138727 after oral dosing of
prasugrel to dogs and was set as 248 (Hagihara et al 2009)
Formation of R-95913 in dog and human intestinal S9 The mixture (total volume
2475 μL) in triplicate contained potassium phosphate buffer (7 mM pH 74) an
NADPH generating system containing 25 mM β-NADP 25 mM G-6-P 05 unitsmL
G-6-PDH and 10 mM MgCl2 5 mM glutathione and dog or human intestinal S9 (10 mg
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proteinmL each) The mixture was preincubated at 37degC for 5 min and 25 μL of
prasugrel (final concentration 250 μM) was added to the mixture which was incubated
at 37degC for 0 5 15 30 and 60 min Ninety μL of ethanol was added to 30 μL of the
reaction mixture to stop the reaction at each time point and the mixture was centrifuged
(15000g 3 min 4degC) Twenty five μL of the supernatant was injected into the HPLC
system HPLC was performed using YMC-ODS A-302 (46 mm id times150 mm)
pumped at flow rate of 05 mLmin with a mobile phase consisting of acetonitrile
isopropyl alcohol distilled water trifluoroacetic acid (51283001 vvvv)
Absorption of the column effluent was monitored at 220 nm to detect the metabolites
Lower limit of quantification for R-95913 was 1 μM
Formation of R-138727 in human and dog intestinal and dog liver microsomes The
mixture (total volume 110 μL) in triplicate contained potassium phosphate buffer (43
mM pH 74) an NADPH generating system (13 mM NADP+ 33 mM
glucose-6-phosphate 04 unitsmL glucose-6-phosphate dehydrogenase and 33 mM
MgCl2) 5 mM glutathione and human or dog intestinal or dog liver microsomes (1 mg
proteinmL each) The mixture was preincubated at 37degC for 5 min and 11 μL of
R-95913 (final concentrations ranging from 16 to 200 μM) was added to the mixture
After incubation at 37degC for 15 min 200 μL of 5 mM methoxyphenacyl bromide
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solution and 100 μL of the IS solution (100 ngmL R-135766) in acetonitrile were added
to 100 μL of the reaction mixture to stop the reaction and the mixture was left for 10
min at room temperature to derivaitze a thiol moiety of R-138727 The mixture was
extracted by a solid phase extract column (Captiva Varian Inc Palo Alto CA) The
assay of R-138727 was performed following the methods previously reported (Hagihara
et al 2009) Separation of the analytes by HPLC was conducted using an
Alliance2690 Separations Module (Waters Co Milford MA) Mass spectra were
determined using a Quattro LC MSMS system (Micromass Ltd Milford MA) in the
positive ion detection mode using an ESI-interface A lower limit of quantification
was set at 16 nM Data acquisition and analyses were performed using MassLynx
software (Version 40 Micromass Ltd)
Data handling The formation pattern of R-138727 in human or dog intestinal or dog
liver microsomes appeared monophasic in each Eadie-Hofstee plot (data not shown)
Therefore the data were fitted to eq 1 using WinNonlin Professional (version 401
Pharsight Corp)
V = Vmax times S (Km + S) (6)
where S Km and Vmax is the substrate concentration Michaelis-Menten constant and
maximal formation rate respectively The intrinsic clearance (CLint) was calculated as
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a ratio of Vmax to Km These parameters are expressed as meanplusmnSD
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Results
Plasma concentrations and AUC0-2hs after intraduodenal administration of
prasugrel to cannulated dogs The plasma concentrations of R-95913 and R-138727
were determined by LC-MS after intraduodenal administration of prasugrel to the portal
and hepatic vein cannulated dogs at a dose of 1 mgkg Plasma concentrations of
prasugrel were not analyzed as prasugrel was not detected unmodified even in the portal
vein in a previous study (Hagihara et al 2009) The plasma concentration-time
profiles of R-95913 and R-138727 are shown in Figures 2 and 3 respectively The
AUC0-2h value of R-95913 in the portal vein (525plusmn234 nghmL) was much higher than
those in hepatic and systemic veins (32plusmn17 and 17plusmn8 nghmL respectively) The
AUC0-2h values of R-138727 in the portal hepatic and systemic veins were 564plusmn187
nghmL 529plusmn136 nghmL and 495plusmn233 nghmL respectively The bimodal peaks
were detected in the plasma concentration-time profiles of R-95913 (Figure 2) This
may indicate reabsorption of R-95913 from the intestine as in the case of rats which
showed enterohepatic circulation of 14C-prasugrel (Hagihara et al 2007)
Availability of R-95913 and R-138727 in the intestine and the liver The FaFg prasrarr
913 and Fh 913 values were 080plusmn023 and 008plusmn006 respectively (Table 1) The
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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INTRODUCTION
Prasugrel (Effientreg in the US and Efientreg in EU) clopidogrel (PlavixregIscoverreg) and
ticlopidine (TiclidTM) are thienopyridine antiplatelet agents Prasugrel is indicated to
reduce the rate of thrombotic cardiovascular events and stent thrombosis in patients
with acute coronary syndrome that are undergoing percutaneous coronary intervention
(Wiviott et al 2007 Effient package insert) The thienopyridines are prodrugs that
are converted in vivo to their pharmacologically active metabolites possessing a thiol
group via a corresponding thiolactone metabolite (Farid et al 2010) In clinical trials
prasugrel achieved greater and faster antiplatelet effect than clopidogrel (Payne et al
2007 Wallentin et al 2008) Such responses to prasugrel are attributed to higher and
faster exposure to its active metabolite R-138727 than clopidogrelrsquos (Sugidachi et al
2007 Ernest et al 2008) Prasugrel is rapidly hydrolyzed to a thiolactone
intermediate R-95913 mainly by human carboxylesterase 2 during absorption through
the gastrointestinal tract (Williams et al 2008) R-95913 is metabolized to R-138727
by cytochrome P450 (CYP) isoforms and the main contributors are CYP3A4 and
CYP2B6 (38-70 and 2-36 respectively) with smaller contributions by CYP2C9
and CYP2C19 (14-19 and 8-11 respectively Rehmel et al 2006) CYP3A5 is as
effective as CYP3A4 in converting R-95913 to R-138727 (Baker et al 2008)
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indicating that CYP3A is a key isoform for R-138727 formation Considering that
CYP3A represents about 80 of the intestinal CYP forms (Paine et al 2006) a large
proportion of R-138727 could be formed during first-pass metabolism in the intestine
We previously detected R-138727 in the portal vein after intraduodenal administration
of prasugrel to rats (Hagihara et al 2009) possibly indicating the formation of
R-138727 in the intestine However that does not prove unequivocally the intestinal
contribution because the appearance of R-138727 in the portal vein was slow and could
have been derived from the circulation after formation in the liver (Hagihara et al
2009)
In this study we determined quantitatively the contribution ratio of the intestine and
liver to the formation of the intermediate R-95913 and the active metabolite R-138727
from prasugrel in dogs
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Materials and Methods
Materials Prasugrel prasugrel thiolactone (R-95913) and prasugrel active metabolite
(R-138727) shown in Figure 1 were synthesized by Ube Industries Ltd (Ube Japan)
Phenacetine and NN-dimethylacetamide were purchased from Sigma-Aldrich (St
Louis MO) Polyethylene glycol 400 was obtained from Wako Pure Chemical
Industries Ltd (Osaka Japan) Methoxyphenacyl bromide was obtained from Tokyo
Kasei Kogyo Co Ltd (Tokyo Japan) Human intestinal microsomes dog intestinal
microsomes and dog liver microsomes were purchased from Tissue Transformation
Technologies Inc (Edison NJ) Biopredic International (Rennes France) and
XenoTech (Lenexa KS) respectively All other chemicals and reagents were
commercially available and of the highest grade
Preparation of dosing formulations Prasugrel was dissolved in 5
NN-dimethylacetamide95 polyethylene glycol 400 (vv) solution at concentrations
of 3 mgmL Solution was prepared immediately before use
Experimental animals Male Beagle dogs (n=6) at ages of 2-3 years were obtained
from Nosan Corporation and were acclimatized in a controlled animal area set at
23plusmn2degC (acceptable range 20-26degC) and relative humidity of 55plusmn5 (acceptable range
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30-70) under a 12-h cycle of lightdark artificial lighting (lighting time 700 AM -
700 PM) Food (DS-A Oriental Yeast Co Ltd Tokyo Japan) and water were
provided freely Dogs weighing 9 to 12 kg that underwent the portal- and
hepatic-vein cannulating surgery were used in the experiments and were fasted for
about 19 h prior to prasugrel administration All animal experiments were carried out
according to the guidelines provided by the Institutional Animal Care and Use
Committee of Daiichi Sankyo Co Ltd
Portal- and hepatic-vein cannulated dogs Under pentobarbital anesthesia the
abdomen was incised on the median line The portal vein was clamped onto the
liver-leaning and intestine-leaning sites of the cannulation point into which the catheter
(Medicut LCV-UK kit single lumen outer diameter 16G length 70 cm Sherwood
Medical Company St Louis MO) was inserted A purse string ligature was applied
and the clamps were released After the portal vein cannulation the same catheter was
also inserted into the hepatic vein and a purse string ligature was applied The
catheters were conjugated with each injection port which was embedded
subcutaneously
Animal experiments From the jugular vein 2 mL of blood was extracted before
dosing The dosing solution of prasugrel was intraduodenally administered to the
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cannulated dogs (n=6) at a dose level of 1 mgkg After about 1 mL of blood drainage
from the portal and hepatic veins via the injection port the syringe was changed to
another one and 1 mL of blood was collected with the heparinized syringe at 1 5 10
15 30 60 and 120 min post-dose The blood in the cannula was pushed back by the
injection of 2 mL of heparin solution in saline (100 unitsmL) At each time point 1
mL of blood was also collected from the jugular vein The blood samples were
immediately centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd Tokyo
Japan) to collect plasma samples A total of 50 μL of plasma was mixed with 100 μL
of acetonitrile and 50 μL of the IS solution (4 μM phenacetin in acetonitrile) and the
mixture was centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd) A
total of 10 μL of the supernatant was injected to LC-MS
Quantitation of R-95913 and R-138727 The quantitation of R-95913 and R-138727
in dog plasma was carried out on an Alliance HPLC system consisting of a 2690
Separations Module (Waters Co Milford MA) coupled to a Quattro LC MS system
(Micromass Ltd Milford MA) with the ESI source in positive ion mode The mobile
phase containing acetonitrile 5 mM ammonium acetate and formic acid (326802
vvv) was applied onto the column A total of 10 μL of each sample was injected onto
a CAPCEL PAK C18 column (5 μm 150 mm times 15 mm Shiseido Co Ltd Tokyo
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Japan) The operating parameters of the MS detector were set as follows capillary
voltage 35 kV ion source temperature 120degC and desolvation temperature 350degC
The detection was performed in the multiple reaction monitoring (MRM) mode The
concentrations of each analyte in the samples were calculated using the computer
software MassLynx (Version 34 Micromass Ltd)
Pharmacokinetic analysis The pharmacokinetic parameters were calculated using the
computer program WinNonlin Professional (ver 401 Pharsight Corp Mountain View
CA) based on the non-compartmental method The area under the plasma
concentration-time curve up to 2 h (AUC0-2h) was calculated by the trapezoidal method
and are expressed as the meanplusmnSD
Calculation of availabililty of R-95913 The availability of R-95913 was calculated
using the following equations
Fa Fg prasrarr913 = Qp Rb (AUCpor913 - AUCsys913) Dose (1)
Fh 913 = AUChep913 AUCpor913 (2)
where Fa Fg prasrarr913 and Fh 913 are fraction of intestinal absorption of prasugrel fraction
of prasugrel hydrolysis to R-95913 in the intestine and availability of R-95913 in the
liver respectively AUCpor913 AUChep913 and AUCsys913 indicate the AUC0-2h values
of R-95913 in the portal hepatic and systemic veins respectively Qp is portal blood
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flow and Rb is the bloodplasma concentration ratio Fa Qp and Rb are set as 0968
(Hagihara et al 2007) 521 mLmin (Hoshino et al 1986) and 0597 (In-house data
Daiichi Sankyo Co Ltd) respectively
Calculation of availabililty of R-138727 The availability of R-138727 was calculated
using the following equations
Fa Fg prasrarr727 = Qp Rb (AUCpor727 - AUCsys727) Dose (3)
Fm 913rarr727 = (AUChep727 - AUCpor727 Fh 727 ) AUCpor913 (4)
Fa Fg prasrarr727 Fh 727 + Fa Fg prasrarr913 Fm 913rarr727 = Relative BA (5)
where Fg prasrarr727 Fm 913rarr727 and Fh 727 are fraction of prasugrel conversion to R-138727
in the intestine fraction of R-95913 conversion to R-138727 in the liver and availability
of R-138727 in the liver respectively AUCpor727 AUChep727 and AUCsys727 indicate
the AUC0-2h values of R-138727 in the portal hepatic and systemic veins respectively
Relative BA means the relative bioavailability of R-138727 after oral dosing of
prasugrel to dogs and was set as 248 (Hagihara et al 2009)
Formation of R-95913 in dog and human intestinal S9 The mixture (total volume
2475 μL) in triplicate contained potassium phosphate buffer (7 mM pH 74) an
NADPH generating system containing 25 mM β-NADP 25 mM G-6-P 05 unitsmL
G-6-PDH and 10 mM MgCl2 5 mM glutathione and dog or human intestinal S9 (10 mg
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proteinmL each) The mixture was preincubated at 37degC for 5 min and 25 μL of
prasugrel (final concentration 250 μM) was added to the mixture which was incubated
at 37degC for 0 5 15 30 and 60 min Ninety μL of ethanol was added to 30 μL of the
reaction mixture to stop the reaction at each time point and the mixture was centrifuged
(15000g 3 min 4degC) Twenty five μL of the supernatant was injected into the HPLC
system HPLC was performed using YMC-ODS A-302 (46 mm id times150 mm)
pumped at flow rate of 05 mLmin with a mobile phase consisting of acetonitrile
isopropyl alcohol distilled water trifluoroacetic acid (51283001 vvvv)
Absorption of the column effluent was monitored at 220 nm to detect the metabolites
Lower limit of quantification for R-95913 was 1 μM
Formation of R-138727 in human and dog intestinal and dog liver microsomes The
mixture (total volume 110 μL) in triplicate contained potassium phosphate buffer (43
mM pH 74) an NADPH generating system (13 mM NADP+ 33 mM
glucose-6-phosphate 04 unitsmL glucose-6-phosphate dehydrogenase and 33 mM
MgCl2) 5 mM glutathione and human or dog intestinal or dog liver microsomes (1 mg
proteinmL each) The mixture was preincubated at 37degC for 5 min and 11 μL of
R-95913 (final concentrations ranging from 16 to 200 μM) was added to the mixture
After incubation at 37degC for 15 min 200 μL of 5 mM methoxyphenacyl bromide
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solution and 100 μL of the IS solution (100 ngmL R-135766) in acetonitrile were added
to 100 μL of the reaction mixture to stop the reaction and the mixture was left for 10
min at room temperature to derivaitze a thiol moiety of R-138727 The mixture was
extracted by a solid phase extract column (Captiva Varian Inc Palo Alto CA) The
assay of R-138727 was performed following the methods previously reported (Hagihara
et al 2009) Separation of the analytes by HPLC was conducted using an
Alliance2690 Separations Module (Waters Co Milford MA) Mass spectra were
determined using a Quattro LC MSMS system (Micromass Ltd Milford MA) in the
positive ion detection mode using an ESI-interface A lower limit of quantification
was set at 16 nM Data acquisition and analyses were performed using MassLynx
software (Version 40 Micromass Ltd)
Data handling The formation pattern of R-138727 in human or dog intestinal or dog
liver microsomes appeared monophasic in each Eadie-Hofstee plot (data not shown)
Therefore the data were fitted to eq 1 using WinNonlin Professional (version 401
Pharsight Corp)
V = Vmax times S (Km + S) (6)
where S Km and Vmax is the substrate concentration Michaelis-Menten constant and
maximal formation rate respectively The intrinsic clearance (CLint) was calculated as
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a ratio of Vmax to Km These parameters are expressed as meanplusmnSD
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Results
Plasma concentrations and AUC0-2hs after intraduodenal administration of
prasugrel to cannulated dogs The plasma concentrations of R-95913 and R-138727
were determined by LC-MS after intraduodenal administration of prasugrel to the portal
and hepatic vein cannulated dogs at a dose of 1 mgkg Plasma concentrations of
prasugrel were not analyzed as prasugrel was not detected unmodified even in the portal
vein in a previous study (Hagihara et al 2009) The plasma concentration-time
profiles of R-95913 and R-138727 are shown in Figures 2 and 3 respectively The
AUC0-2h value of R-95913 in the portal vein (525plusmn234 nghmL) was much higher than
those in hepatic and systemic veins (32plusmn17 and 17plusmn8 nghmL respectively) The
AUC0-2h values of R-138727 in the portal hepatic and systemic veins were 564plusmn187
nghmL 529plusmn136 nghmL and 495plusmn233 nghmL respectively The bimodal peaks
were detected in the plasma concentration-time profiles of R-95913 (Figure 2) This
may indicate reabsorption of R-95913 from the intestine as in the case of rats which
showed enterohepatic circulation of 14C-prasugrel (Hagihara et al 2007)
Availability of R-95913 and R-138727 in the intestine and the liver The FaFg prasrarr
913 and Fh 913 values were 080plusmn023 and 008plusmn006 respectively (Table 1) The
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
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Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
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Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
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This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
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Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
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Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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indicating that CYP3A is a key isoform for R-138727 formation Considering that
CYP3A represents about 80 of the intestinal CYP forms (Paine et al 2006) a large
proportion of R-138727 could be formed during first-pass metabolism in the intestine
We previously detected R-138727 in the portal vein after intraduodenal administration
of prasugrel to rats (Hagihara et al 2009) possibly indicating the formation of
R-138727 in the intestine However that does not prove unequivocally the intestinal
contribution because the appearance of R-138727 in the portal vein was slow and could
have been derived from the circulation after formation in the liver (Hagihara et al
2009)
In this study we determined quantitatively the contribution ratio of the intestine and
liver to the formation of the intermediate R-95913 and the active metabolite R-138727
from prasugrel in dogs
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Materials and Methods
Materials Prasugrel prasugrel thiolactone (R-95913) and prasugrel active metabolite
(R-138727) shown in Figure 1 were synthesized by Ube Industries Ltd (Ube Japan)
Phenacetine and NN-dimethylacetamide were purchased from Sigma-Aldrich (St
Louis MO) Polyethylene glycol 400 was obtained from Wako Pure Chemical
Industries Ltd (Osaka Japan) Methoxyphenacyl bromide was obtained from Tokyo
Kasei Kogyo Co Ltd (Tokyo Japan) Human intestinal microsomes dog intestinal
microsomes and dog liver microsomes were purchased from Tissue Transformation
Technologies Inc (Edison NJ) Biopredic International (Rennes France) and
XenoTech (Lenexa KS) respectively All other chemicals and reagents were
commercially available and of the highest grade
Preparation of dosing formulations Prasugrel was dissolved in 5
NN-dimethylacetamide95 polyethylene glycol 400 (vv) solution at concentrations
of 3 mgmL Solution was prepared immediately before use
Experimental animals Male Beagle dogs (n=6) at ages of 2-3 years were obtained
from Nosan Corporation and were acclimatized in a controlled animal area set at
23plusmn2degC (acceptable range 20-26degC) and relative humidity of 55plusmn5 (acceptable range
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30-70) under a 12-h cycle of lightdark artificial lighting (lighting time 700 AM -
700 PM) Food (DS-A Oriental Yeast Co Ltd Tokyo Japan) and water were
provided freely Dogs weighing 9 to 12 kg that underwent the portal- and
hepatic-vein cannulating surgery were used in the experiments and were fasted for
about 19 h prior to prasugrel administration All animal experiments were carried out
according to the guidelines provided by the Institutional Animal Care and Use
Committee of Daiichi Sankyo Co Ltd
Portal- and hepatic-vein cannulated dogs Under pentobarbital anesthesia the
abdomen was incised on the median line The portal vein was clamped onto the
liver-leaning and intestine-leaning sites of the cannulation point into which the catheter
(Medicut LCV-UK kit single lumen outer diameter 16G length 70 cm Sherwood
Medical Company St Louis MO) was inserted A purse string ligature was applied
and the clamps were released After the portal vein cannulation the same catheter was
also inserted into the hepatic vein and a purse string ligature was applied The
catheters were conjugated with each injection port which was embedded
subcutaneously
Animal experiments From the jugular vein 2 mL of blood was extracted before
dosing The dosing solution of prasugrel was intraduodenally administered to the
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cannulated dogs (n=6) at a dose level of 1 mgkg After about 1 mL of blood drainage
from the portal and hepatic veins via the injection port the syringe was changed to
another one and 1 mL of blood was collected with the heparinized syringe at 1 5 10
15 30 60 and 120 min post-dose The blood in the cannula was pushed back by the
injection of 2 mL of heparin solution in saline (100 unitsmL) At each time point 1
mL of blood was also collected from the jugular vein The blood samples were
immediately centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd Tokyo
Japan) to collect plasma samples A total of 50 μL of plasma was mixed with 100 μL
of acetonitrile and 50 μL of the IS solution (4 μM phenacetin in acetonitrile) and the
mixture was centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd) A
total of 10 μL of the supernatant was injected to LC-MS
Quantitation of R-95913 and R-138727 The quantitation of R-95913 and R-138727
in dog plasma was carried out on an Alliance HPLC system consisting of a 2690
Separations Module (Waters Co Milford MA) coupled to a Quattro LC MS system
(Micromass Ltd Milford MA) with the ESI source in positive ion mode The mobile
phase containing acetonitrile 5 mM ammonium acetate and formic acid (326802
vvv) was applied onto the column A total of 10 μL of each sample was injected onto
a CAPCEL PAK C18 column (5 μm 150 mm times 15 mm Shiseido Co Ltd Tokyo
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Japan) The operating parameters of the MS detector were set as follows capillary
voltage 35 kV ion source temperature 120degC and desolvation temperature 350degC
The detection was performed in the multiple reaction monitoring (MRM) mode The
concentrations of each analyte in the samples were calculated using the computer
software MassLynx (Version 34 Micromass Ltd)
Pharmacokinetic analysis The pharmacokinetic parameters were calculated using the
computer program WinNonlin Professional (ver 401 Pharsight Corp Mountain View
CA) based on the non-compartmental method The area under the plasma
concentration-time curve up to 2 h (AUC0-2h) was calculated by the trapezoidal method
and are expressed as the meanplusmnSD
Calculation of availabililty of R-95913 The availability of R-95913 was calculated
using the following equations
Fa Fg prasrarr913 = Qp Rb (AUCpor913 - AUCsys913) Dose (1)
Fh 913 = AUChep913 AUCpor913 (2)
where Fa Fg prasrarr913 and Fh 913 are fraction of intestinal absorption of prasugrel fraction
of prasugrel hydrolysis to R-95913 in the intestine and availability of R-95913 in the
liver respectively AUCpor913 AUChep913 and AUCsys913 indicate the AUC0-2h values
of R-95913 in the portal hepatic and systemic veins respectively Qp is portal blood
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flow and Rb is the bloodplasma concentration ratio Fa Qp and Rb are set as 0968
(Hagihara et al 2007) 521 mLmin (Hoshino et al 1986) and 0597 (In-house data
Daiichi Sankyo Co Ltd) respectively
Calculation of availabililty of R-138727 The availability of R-138727 was calculated
using the following equations
Fa Fg prasrarr727 = Qp Rb (AUCpor727 - AUCsys727) Dose (3)
Fm 913rarr727 = (AUChep727 - AUCpor727 Fh 727 ) AUCpor913 (4)
Fa Fg prasrarr727 Fh 727 + Fa Fg prasrarr913 Fm 913rarr727 = Relative BA (5)
where Fg prasrarr727 Fm 913rarr727 and Fh 727 are fraction of prasugrel conversion to R-138727
in the intestine fraction of R-95913 conversion to R-138727 in the liver and availability
of R-138727 in the liver respectively AUCpor727 AUChep727 and AUCsys727 indicate
the AUC0-2h values of R-138727 in the portal hepatic and systemic veins respectively
Relative BA means the relative bioavailability of R-138727 after oral dosing of
prasugrel to dogs and was set as 248 (Hagihara et al 2009)
Formation of R-95913 in dog and human intestinal S9 The mixture (total volume
2475 μL) in triplicate contained potassium phosphate buffer (7 mM pH 74) an
NADPH generating system containing 25 mM β-NADP 25 mM G-6-P 05 unitsmL
G-6-PDH and 10 mM MgCl2 5 mM glutathione and dog or human intestinal S9 (10 mg
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proteinmL each) The mixture was preincubated at 37degC for 5 min and 25 μL of
prasugrel (final concentration 250 μM) was added to the mixture which was incubated
at 37degC for 0 5 15 30 and 60 min Ninety μL of ethanol was added to 30 μL of the
reaction mixture to stop the reaction at each time point and the mixture was centrifuged
(15000g 3 min 4degC) Twenty five μL of the supernatant was injected into the HPLC
system HPLC was performed using YMC-ODS A-302 (46 mm id times150 mm)
pumped at flow rate of 05 mLmin with a mobile phase consisting of acetonitrile
isopropyl alcohol distilled water trifluoroacetic acid (51283001 vvvv)
Absorption of the column effluent was monitored at 220 nm to detect the metabolites
Lower limit of quantification for R-95913 was 1 μM
Formation of R-138727 in human and dog intestinal and dog liver microsomes The
mixture (total volume 110 μL) in triplicate contained potassium phosphate buffer (43
mM pH 74) an NADPH generating system (13 mM NADP+ 33 mM
glucose-6-phosphate 04 unitsmL glucose-6-phosphate dehydrogenase and 33 mM
MgCl2) 5 mM glutathione and human or dog intestinal or dog liver microsomes (1 mg
proteinmL each) The mixture was preincubated at 37degC for 5 min and 11 μL of
R-95913 (final concentrations ranging from 16 to 200 μM) was added to the mixture
After incubation at 37degC for 15 min 200 μL of 5 mM methoxyphenacyl bromide
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solution and 100 μL of the IS solution (100 ngmL R-135766) in acetonitrile were added
to 100 μL of the reaction mixture to stop the reaction and the mixture was left for 10
min at room temperature to derivaitze a thiol moiety of R-138727 The mixture was
extracted by a solid phase extract column (Captiva Varian Inc Palo Alto CA) The
assay of R-138727 was performed following the methods previously reported (Hagihara
et al 2009) Separation of the analytes by HPLC was conducted using an
Alliance2690 Separations Module (Waters Co Milford MA) Mass spectra were
determined using a Quattro LC MSMS system (Micromass Ltd Milford MA) in the
positive ion detection mode using an ESI-interface A lower limit of quantification
was set at 16 nM Data acquisition and analyses were performed using MassLynx
software (Version 40 Micromass Ltd)
Data handling The formation pattern of R-138727 in human or dog intestinal or dog
liver microsomes appeared monophasic in each Eadie-Hofstee plot (data not shown)
Therefore the data were fitted to eq 1 using WinNonlin Professional (version 401
Pharsight Corp)
V = Vmax times S (Km + S) (6)
where S Km and Vmax is the substrate concentration Michaelis-Menten constant and
maximal formation rate respectively The intrinsic clearance (CLint) was calculated as
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a ratio of Vmax to Km These parameters are expressed as meanplusmnSD
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Results
Plasma concentrations and AUC0-2hs after intraduodenal administration of
prasugrel to cannulated dogs The plasma concentrations of R-95913 and R-138727
were determined by LC-MS after intraduodenal administration of prasugrel to the portal
and hepatic vein cannulated dogs at a dose of 1 mgkg Plasma concentrations of
prasugrel were not analyzed as prasugrel was not detected unmodified even in the portal
vein in a previous study (Hagihara et al 2009) The plasma concentration-time
profiles of R-95913 and R-138727 are shown in Figures 2 and 3 respectively The
AUC0-2h value of R-95913 in the portal vein (525plusmn234 nghmL) was much higher than
those in hepatic and systemic veins (32plusmn17 and 17plusmn8 nghmL respectively) The
AUC0-2h values of R-138727 in the portal hepatic and systemic veins were 564plusmn187
nghmL 529plusmn136 nghmL and 495plusmn233 nghmL respectively The bimodal peaks
were detected in the plasma concentration-time profiles of R-95913 (Figure 2) This
may indicate reabsorption of R-95913 from the intestine as in the case of rats which
showed enterohepatic circulation of 14C-prasugrel (Hagihara et al 2007)
Availability of R-95913 and R-138727 in the intestine and the liver The FaFg prasrarr
913 and Fh 913 values were 080plusmn023 and 008plusmn006 respectively (Table 1) The
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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Materials and Methods
Materials Prasugrel prasugrel thiolactone (R-95913) and prasugrel active metabolite
(R-138727) shown in Figure 1 were synthesized by Ube Industries Ltd (Ube Japan)
Phenacetine and NN-dimethylacetamide were purchased from Sigma-Aldrich (St
Louis MO) Polyethylene glycol 400 was obtained from Wako Pure Chemical
Industries Ltd (Osaka Japan) Methoxyphenacyl bromide was obtained from Tokyo
Kasei Kogyo Co Ltd (Tokyo Japan) Human intestinal microsomes dog intestinal
microsomes and dog liver microsomes were purchased from Tissue Transformation
Technologies Inc (Edison NJ) Biopredic International (Rennes France) and
XenoTech (Lenexa KS) respectively All other chemicals and reagents were
commercially available and of the highest grade
Preparation of dosing formulations Prasugrel was dissolved in 5
NN-dimethylacetamide95 polyethylene glycol 400 (vv) solution at concentrations
of 3 mgmL Solution was prepared immediately before use
Experimental animals Male Beagle dogs (n=6) at ages of 2-3 years were obtained
from Nosan Corporation and were acclimatized in a controlled animal area set at
23plusmn2degC (acceptable range 20-26degC) and relative humidity of 55plusmn5 (acceptable range
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30-70) under a 12-h cycle of lightdark artificial lighting (lighting time 700 AM -
700 PM) Food (DS-A Oriental Yeast Co Ltd Tokyo Japan) and water were
provided freely Dogs weighing 9 to 12 kg that underwent the portal- and
hepatic-vein cannulating surgery were used in the experiments and were fasted for
about 19 h prior to prasugrel administration All animal experiments were carried out
according to the guidelines provided by the Institutional Animal Care and Use
Committee of Daiichi Sankyo Co Ltd
Portal- and hepatic-vein cannulated dogs Under pentobarbital anesthesia the
abdomen was incised on the median line The portal vein was clamped onto the
liver-leaning and intestine-leaning sites of the cannulation point into which the catheter
(Medicut LCV-UK kit single lumen outer diameter 16G length 70 cm Sherwood
Medical Company St Louis MO) was inserted A purse string ligature was applied
and the clamps were released After the portal vein cannulation the same catheter was
also inserted into the hepatic vein and a purse string ligature was applied The
catheters were conjugated with each injection port which was embedded
subcutaneously
Animal experiments From the jugular vein 2 mL of blood was extracted before
dosing The dosing solution of prasugrel was intraduodenally administered to the
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cannulated dogs (n=6) at a dose level of 1 mgkg After about 1 mL of blood drainage
from the portal and hepatic veins via the injection port the syringe was changed to
another one and 1 mL of blood was collected with the heparinized syringe at 1 5 10
15 30 60 and 120 min post-dose The blood in the cannula was pushed back by the
injection of 2 mL of heparin solution in saline (100 unitsmL) At each time point 1
mL of blood was also collected from the jugular vein The blood samples were
immediately centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd Tokyo
Japan) to collect plasma samples A total of 50 μL of plasma was mixed with 100 μL
of acetonitrile and 50 μL of the IS solution (4 μM phenacetin in acetonitrile) and the
mixture was centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd) A
total of 10 μL of the supernatant was injected to LC-MS
Quantitation of R-95913 and R-138727 The quantitation of R-95913 and R-138727
in dog plasma was carried out on an Alliance HPLC system consisting of a 2690
Separations Module (Waters Co Milford MA) coupled to a Quattro LC MS system
(Micromass Ltd Milford MA) with the ESI source in positive ion mode The mobile
phase containing acetonitrile 5 mM ammonium acetate and formic acid (326802
vvv) was applied onto the column A total of 10 μL of each sample was injected onto
a CAPCEL PAK C18 column (5 μm 150 mm times 15 mm Shiseido Co Ltd Tokyo
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Japan) The operating parameters of the MS detector were set as follows capillary
voltage 35 kV ion source temperature 120degC and desolvation temperature 350degC
The detection was performed in the multiple reaction monitoring (MRM) mode The
concentrations of each analyte in the samples were calculated using the computer
software MassLynx (Version 34 Micromass Ltd)
Pharmacokinetic analysis The pharmacokinetic parameters were calculated using the
computer program WinNonlin Professional (ver 401 Pharsight Corp Mountain View
CA) based on the non-compartmental method The area under the plasma
concentration-time curve up to 2 h (AUC0-2h) was calculated by the trapezoidal method
and are expressed as the meanplusmnSD
Calculation of availabililty of R-95913 The availability of R-95913 was calculated
using the following equations
Fa Fg prasrarr913 = Qp Rb (AUCpor913 - AUCsys913) Dose (1)
Fh 913 = AUChep913 AUCpor913 (2)
where Fa Fg prasrarr913 and Fh 913 are fraction of intestinal absorption of prasugrel fraction
of prasugrel hydrolysis to R-95913 in the intestine and availability of R-95913 in the
liver respectively AUCpor913 AUChep913 and AUCsys913 indicate the AUC0-2h values
of R-95913 in the portal hepatic and systemic veins respectively Qp is portal blood
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flow and Rb is the bloodplasma concentration ratio Fa Qp and Rb are set as 0968
(Hagihara et al 2007) 521 mLmin (Hoshino et al 1986) and 0597 (In-house data
Daiichi Sankyo Co Ltd) respectively
Calculation of availabililty of R-138727 The availability of R-138727 was calculated
using the following equations
Fa Fg prasrarr727 = Qp Rb (AUCpor727 - AUCsys727) Dose (3)
Fm 913rarr727 = (AUChep727 - AUCpor727 Fh 727 ) AUCpor913 (4)
Fa Fg prasrarr727 Fh 727 + Fa Fg prasrarr913 Fm 913rarr727 = Relative BA (5)
where Fg prasrarr727 Fm 913rarr727 and Fh 727 are fraction of prasugrel conversion to R-138727
in the intestine fraction of R-95913 conversion to R-138727 in the liver and availability
of R-138727 in the liver respectively AUCpor727 AUChep727 and AUCsys727 indicate
the AUC0-2h values of R-138727 in the portal hepatic and systemic veins respectively
Relative BA means the relative bioavailability of R-138727 after oral dosing of
prasugrel to dogs and was set as 248 (Hagihara et al 2009)
Formation of R-95913 in dog and human intestinal S9 The mixture (total volume
2475 μL) in triplicate contained potassium phosphate buffer (7 mM pH 74) an
NADPH generating system containing 25 mM β-NADP 25 mM G-6-P 05 unitsmL
G-6-PDH and 10 mM MgCl2 5 mM glutathione and dog or human intestinal S9 (10 mg
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proteinmL each) The mixture was preincubated at 37degC for 5 min and 25 μL of
prasugrel (final concentration 250 μM) was added to the mixture which was incubated
at 37degC for 0 5 15 30 and 60 min Ninety μL of ethanol was added to 30 μL of the
reaction mixture to stop the reaction at each time point and the mixture was centrifuged
(15000g 3 min 4degC) Twenty five μL of the supernatant was injected into the HPLC
system HPLC was performed using YMC-ODS A-302 (46 mm id times150 mm)
pumped at flow rate of 05 mLmin with a mobile phase consisting of acetonitrile
isopropyl alcohol distilled water trifluoroacetic acid (51283001 vvvv)
Absorption of the column effluent was monitored at 220 nm to detect the metabolites
Lower limit of quantification for R-95913 was 1 μM
Formation of R-138727 in human and dog intestinal and dog liver microsomes The
mixture (total volume 110 μL) in triplicate contained potassium phosphate buffer (43
mM pH 74) an NADPH generating system (13 mM NADP+ 33 mM
glucose-6-phosphate 04 unitsmL glucose-6-phosphate dehydrogenase and 33 mM
MgCl2) 5 mM glutathione and human or dog intestinal or dog liver microsomes (1 mg
proteinmL each) The mixture was preincubated at 37degC for 5 min and 11 μL of
R-95913 (final concentrations ranging from 16 to 200 μM) was added to the mixture
After incubation at 37degC for 15 min 200 μL of 5 mM methoxyphenacyl bromide
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solution and 100 μL of the IS solution (100 ngmL R-135766) in acetonitrile were added
to 100 μL of the reaction mixture to stop the reaction and the mixture was left for 10
min at room temperature to derivaitze a thiol moiety of R-138727 The mixture was
extracted by a solid phase extract column (Captiva Varian Inc Palo Alto CA) The
assay of R-138727 was performed following the methods previously reported (Hagihara
et al 2009) Separation of the analytes by HPLC was conducted using an
Alliance2690 Separations Module (Waters Co Milford MA) Mass spectra were
determined using a Quattro LC MSMS system (Micromass Ltd Milford MA) in the
positive ion detection mode using an ESI-interface A lower limit of quantification
was set at 16 nM Data acquisition and analyses were performed using MassLynx
software (Version 40 Micromass Ltd)
Data handling The formation pattern of R-138727 in human or dog intestinal or dog
liver microsomes appeared monophasic in each Eadie-Hofstee plot (data not shown)
Therefore the data were fitted to eq 1 using WinNonlin Professional (version 401
Pharsight Corp)
V = Vmax times S (Km + S) (6)
where S Km and Vmax is the substrate concentration Michaelis-Menten constant and
maximal formation rate respectively The intrinsic clearance (CLint) was calculated as
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a ratio of Vmax to Km These parameters are expressed as meanplusmnSD
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Results
Plasma concentrations and AUC0-2hs after intraduodenal administration of
prasugrel to cannulated dogs The plasma concentrations of R-95913 and R-138727
were determined by LC-MS after intraduodenal administration of prasugrel to the portal
and hepatic vein cannulated dogs at a dose of 1 mgkg Plasma concentrations of
prasugrel were not analyzed as prasugrel was not detected unmodified even in the portal
vein in a previous study (Hagihara et al 2009) The plasma concentration-time
profiles of R-95913 and R-138727 are shown in Figures 2 and 3 respectively The
AUC0-2h value of R-95913 in the portal vein (525plusmn234 nghmL) was much higher than
those in hepatic and systemic veins (32plusmn17 and 17plusmn8 nghmL respectively) The
AUC0-2h values of R-138727 in the portal hepatic and systemic veins were 564plusmn187
nghmL 529plusmn136 nghmL and 495plusmn233 nghmL respectively The bimodal peaks
were detected in the plasma concentration-time profiles of R-95913 (Figure 2) This
may indicate reabsorption of R-95913 from the intestine as in the case of rats which
showed enterohepatic circulation of 14C-prasugrel (Hagihara et al 2007)
Availability of R-95913 and R-138727 in the intestine and the liver The FaFg prasrarr
913 and Fh 913 values were 080plusmn023 and 008plusmn006 respectively (Table 1) The
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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30-70) under a 12-h cycle of lightdark artificial lighting (lighting time 700 AM -
700 PM) Food (DS-A Oriental Yeast Co Ltd Tokyo Japan) and water were
provided freely Dogs weighing 9 to 12 kg that underwent the portal- and
hepatic-vein cannulating surgery were used in the experiments and were fasted for
about 19 h prior to prasugrel administration All animal experiments were carried out
according to the guidelines provided by the Institutional Animal Care and Use
Committee of Daiichi Sankyo Co Ltd
Portal- and hepatic-vein cannulated dogs Under pentobarbital anesthesia the
abdomen was incised on the median line The portal vein was clamped onto the
liver-leaning and intestine-leaning sites of the cannulation point into which the catheter
(Medicut LCV-UK kit single lumen outer diameter 16G length 70 cm Sherwood
Medical Company St Louis MO) was inserted A purse string ligature was applied
and the clamps were released After the portal vein cannulation the same catheter was
also inserted into the hepatic vein and a purse string ligature was applied The
catheters were conjugated with each injection port which was embedded
subcutaneously
Animal experiments From the jugular vein 2 mL of blood was extracted before
dosing The dosing solution of prasugrel was intraduodenally administered to the
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cannulated dogs (n=6) at a dose level of 1 mgkg After about 1 mL of blood drainage
from the portal and hepatic veins via the injection port the syringe was changed to
another one and 1 mL of blood was collected with the heparinized syringe at 1 5 10
15 30 60 and 120 min post-dose The blood in the cannula was pushed back by the
injection of 2 mL of heparin solution in saline (100 unitsmL) At each time point 1
mL of blood was also collected from the jugular vein The blood samples were
immediately centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd Tokyo
Japan) to collect plasma samples A total of 50 μL of plasma was mixed with 100 μL
of acetonitrile and 50 μL of the IS solution (4 μM phenacetin in acetonitrile) and the
mixture was centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd) A
total of 10 μL of the supernatant was injected to LC-MS
Quantitation of R-95913 and R-138727 The quantitation of R-95913 and R-138727
in dog plasma was carried out on an Alliance HPLC system consisting of a 2690
Separations Module (Waters Co Milford MA) coupled to a Quattro LC MS system
(Micromass Ltd Milford MA) with the ESI source in positive ion mode The mobile
phase containing acetonitrile 5 mM ammonium acetate and formic acid (326802
vvv) was applied onto the column A total of 10 μL of each sample was injected onto
a CAPCEL PAK C18 column (5 μm 150 mm times 15 mm Shiseido Co Ltd Tokyo
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Japan) The operating parameters of the MS detector were set as follows capillary
voltage 35 kV ion source temperature 120degC and desolvation temperature 350degC
The detection was performed in the multiple reaction monitoring (MRM) mode The
concentrations of each analyte in the samples were calculated using the computer
software MassLynx (Version 34 Micromass Ltd)
Pharmacokinetic analysis The pharmacokinetic parameters were calculated using the
computer program WinNonlin Professional (ver 401 Pharsight Corp Mountain View
CA) based on the non-compartmental method The area under the plasma
concentration-time curve up to 2 h (AUC0-2h) was calculated by the trapezoidal method
and are expressed as the meanplusmnSD
Calculation of availabililty of R-95913 The availability of R-95913 was calculated
using the following equations
Fa Fg prasrarr913 = Qp Rb (AUCpor913 - AUCsys913) Dose (1)
Fh 913 = AUChep913 AUCpor913 (2)
where Fa Fg prasrarr913 and Fh 913 are fraction of intestinal absorption of prasugrel fraction
of prasugrel hydrolysis to R-95913 in the intestine and availability of R-95913 in the
liver respectively AUCpor913 AUChep913 and AUCsys913 indicate the AUC0-2h values
of R-95913 in the portal hepatic and systemic veins respectively Qp is portal blood
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flow and Rb is the bloodplasma concentration ratio Fa Qp and Rb are set as 0968
(Hagihara et al 2007) 521 mLmin (Hoshino et al 1986) and 0597 (In-house data
Daiichi Sankyo Co Ltd) respectively
Calculation of availabililty of R-138727 The availability of R-138727 was calculated
using the following equations
Fa Fg prasrarr727 = Qp Rb (AUCpor727 - AUCsys727) Dose (3)
Fm 913rarr727 = (AUChep727 - AUCpor727 Fh 727 ) AUCpor913 (4)
Fa Fg prasrarr727 Fh 727 + Fa Fg prasrarr913 Fm 913rarr727 = Relative BA (5)
where Fg prasrarr727 Fm 913rarr727 and Fh 727 are fraction of prasugrel conversion to R-138727
in the intestine fraction of R-95913 conversion to R-138727 in the liver and availability
of R-138727 in the liver respectively AUCpor727 AUChep727 and AUCsys727 indicate
the AUC0-2h values of R-138727 in the portal hepatic and systemic veins respectively
Relative BA means the relative bioavailability of R-138727 after oral dosing of
prasugrel to dogs and was set as 248 (Hagihara et al 2009)
Formation of R-95913 in dog and human intestinal S9 The mixture (total volume
2475 μL) in triplicate contained potassium phosphate buffer (7 mM pH 74) an
NADPH generating system containing 25 mM β-NADP 25 mM G-6-P 05 unitsmL
G-6-PDH and 10 mM MgCl2 5 mM glutathione and dog or human intestinal S9 (10 mg
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proteinmL each) The mixture was preincubated at 37degC for 5 min and 25 μL of
prasugrel (final concentration 250 μM) was added to the mixture which was incubated
at 37degC for 0 5 15 30 and 60 min Ninety μL of ethanol was added to 30 μL of the
reaction mixture to stop the reaction at each time point and the mixture was centrifuged
(15000g 3 min 4degC) Twenty five μL of the supernatant was injected into the HPLC
system HPLC was performed using YMC-ODS A-302 (46 mm id times150 mm)
pumped at flow rate of 05 mLmin with a mobile phase consisting of acetonitrile
isopropyl alcohol distilled water trifluoroacetic acid (51283001 vvvv)
Absorption of the column effluent was monitored at 220 nm to detect the metabolites
Lower limit of quantification for R-95913 was 1 μM
Formation of R-138727 in human and dog intestinal and dog liver microsomes The
mixture (total volume 110 μL) in triplicate contained potassium phosphate buffer (43
mM pH 74) an NADPH generating system (13 mM NADP+ 33 mM
glucose-6-phosphate 04 unitsmL glucose-6-phosphate dehydrogenase and 33 mM
MgCl2) 5 mM glutathione and human or dog intestinal or dog liver microsomes (1 mg
proteinmL each) The mixture was preincubated at 37degC for 5 min and 11 μL of
R-95913 (final concentrations ranging from 16 to 200 μM) was added to the mixture
After incubation at 37degC for 15 min 200 μL of 5 mM methoxyphenacyl bromide
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12
solution and 100 μL of the IS solution (100 ngmL R-135766) in acetonitrile were added
to 100 μL of the reaction mixture to stop the reaction and the mixture was left for 10
min at room temperature to derivaitze a thiol moiety of R-138727 The mixture was
extracted by a solid phase extract column (Captiva Varian Inc Palo Alto CA) The
assay of R-138727 was performed following the methods previously reported (Hagihara
et al 2009) Separation of the analytes by HPLC was conducted using an
Alliance2690 Separations Module (Waters Co Milford MA) Mass spectra were
determined using a Quattro LC MSMS system (Micromass Ltd Milford MA) in the
positive ion detection mode using an ESI-interface A lower limit of quantification
was set at 16 nM Data acquisition and analyses were performed using MassLynx
software (Version 40 Micromass Ltd)
Data handling The formation pattern of R-138727 in human or dog intestinal or dog
liver microsomes appeared monophasic in each Eadie-Hofstee plot (data not shown)
Therefore the data were fitted to eq 1 using WinNonlin Professional (version 401
Pharsight Corp)
V = Vmax times S (Km + S) (6)
where S Km and Vmax is the substrate concentration Michaelis-Menten constant and
maximal formation rate respectively The intrinsic clearance (CLint) was calculated as
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13
a ratio of Vmax to Km These parameters are expressed as meanplusmnSD
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Results
Plasma concentrations and AUC0-2hs after intraduodenal administration of
prasugrel to cannulated dogs The plasma concentrations of R-95913 and R-138727
were determined by LC-MS after intraduodenal administration of prasugrel to the portal
and hepatic vein cannulated dogs at a dose of 1 mgkg Plasma concentrations of
prasugrel were not analyzed as prasugrel was not detected unmodified even in the portal
vein in a previous study (Hagihara et al 2009) The plasma concentration-time
profiles of R-95913 and R-138727 are shown in Figures 2 and 3 respectively The
AUC0-2h value of R-95913 in the portal vein (525plusmn234 nghmL) was much higher than
those in hepatic and systemic veins (32plusmn17 and 17plusmn8 nghmL respectively) The
AUC0-2h values of R-138727 in the portal hepatic and systemic veins were 564plusmn187
nghmL 529plusmn136 nghmL and 495plusmn233 nghmL respectively The bimodal peaks
were detected in the plasma concentration-time profiles of R-95913 (Figure 2) This
may indicate reabsorption of R-95913 from the intestine as in the case of rats which
showed enterohepatic circulation of 14C-prasugrel (Hagihara et al 2007)
Availability of R-95913 and R-138727 in the intestine and the liver The FaFg prasrarr
913 and Fh 913 values were 080plusmn023 and 008plusmn006 respectively (Table 1) The
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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cannulated dogs (n=6) at a dose level of 1 mgkg After about 1 mL of blood drainage
from the portal and hepatic veins via the injection port the syringe was changed to
another one and 1 mL of blood was collected with the heparinized syringe at 1 5 10
15 30 60 and 120 min post-dose The blood in the cannula was pushed back by the
injection of 2 mL of heparin solution in saline (100 unitsmL) At each time point 1
mL of blood was also collected from the jugular vein The blood samples were
immediately centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd Tokyo
Japan) to collect plasma samples A total of 50 μL of plasma was mixed with 100 μL
of acetonitrile and 50 μL of the IS solution (4 μM phenacetin in acetonitrile) and the
mixture was centrifuged at 14000 rpm for 3 min at 4degC (Hitachi Koki Co Ltd) A
total of 10 μL of the supernatant was injected to LC-MS
Quantitation of R-95913 and R-138727 The quantitation of R-95913 and R-138727
in dog plasma was carried out on an Alliance HPLC system consisting of a 2690
Separations Module (Waters Co Milford MA) coupled to a Quattro LC MS system
(Micromass Ltd Milford MA) with the ESI source in positive ion mode The mobile
phase containing acetonitrile 5 mM ammonium acetate and formic acid (326802
vvv) was applied onto the column A total of 10 μL of each sample was injected onto
a CAPCEL PAK C18 column (5 μm 150 mm times 15 mm Shiseido Co Ltd Tokyo
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Japan) The operating parameters of the MS detector were set as follows capillary
voltage 35 kV ion source temperature 120degC and desolvation temperature 350degC
The detection was performed in the multiple reaction monitoring (MRM) mode The
concentrations of each analyte in the samples were calculated using the computer
software MassLynx (Version 34 Micromass Ltd)
Pharmacokinetic analysis The pharmacokinetic parameters were calculated using the
computer program WinNonlin Professional (ver 401 Pharsight Corp Mountain View
CA) based on the non-compartmental method The area under the plasma
concentration-time curve up to 2 h (AUC0-2h) was calculated by the trapezoidal method
and are expressed as the meanplusmnSD
Calculation of availabililty of R-95913 The availability of R-95913 was calculated
using the following equations
Fa Fg prasrarr913 = Qp Rb (AUCpor913 - AUCsys913) Dose (1)
Fh 913 = AUChep913 AUCpor913 (2)
where Fa Fg prasrarr913 and Fh 913 are fraction of intestinal absorption of prasugrel fraction
of prasugrel hydrolysis to R-95913 in the intestine and availability of R-95913 in the
liver respectively AUCpor913 AUChep913 and AUCsys913 indicate the AUC0-2h values
of R-95913 in the portal hepatic and systemic veins respectively Qp is portal blood
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flow and Rb is the bloodplasma concentration ratio Fa Qp and Rb are set as 0968
(Hagihara et al 2007) 521 mLmin (Hoshino et al 1986) and 0597 (In-house data
Daiichi Sankyo Co Ltd) respectively
Calculation of availabililty of R-138727 The availability of R-138727 was calculated
using the following equations
Fa Fg prasrarr727 = Qp Rb (AUCpor727 - AUCsys727) Dose (3)
Fm 913rarr727 = (AUChep727 - AUCpor727 Fh 727 ) AUCpor913 (4)
Fa Fg prasrarr727 Fh 727 + Fa Fg prasrarr913 Fm 913rarr727 = Relative BA (5)
where Fg prasrarr727 Fm 913rarr727 and Fh 727 are fraction of prasugrel conversion to R-138727
in the intestine fraction of R-95913 conversion to R-138727 in the liver and availability
of R-138727 in the liver respectively AUCpor727 AUChep727 and AUCsys727 indicate
the AUC0-2h values of R-138727 in the portal hepatic and systemic veins respectively
Relative BA means the relative bioavailability of R-138727 after oral dosing of
prasugrel to dogs and was set as 248 (Hagihara et al 2009)
Formation of R-95913 in dog and human intestinal S9 The mixture (total volume
2475 μL) in triplicate contained potassium phosphate buffer (7 mM pH 74) an
NADPH generating system containing 25 mM β-NADP 25 mM G-6-P 05 unitsmL
G-6-PDH and 10 mM MgCl2 5 mM glutathione and dog or human intestinal S9 (10 mg
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proteinmL each) The mixture was preincubated at 37degC for 5 min and 25 μL of
prasugrel (final concentration 250 μM) was added to the mixture which was incubated
at 37degC for 0 5 15 30 and 60 min Ninety μL of ethanol was added to 30 μL of the
reaction mixture to stop the reaction at each time point and the mixture was centrifuged
(15000g 3 min 4degC) Twenty five μL of the supernatant was injected into the HPLC
system HPLC was performed using YMC-ODS A-302 (46 mm id times150 mm)
pumped at flow rate of 05 mLmin with a mobile phase consisting of acetonitrile
isopropyl alcohol distilled water trifluoroacetic acid (51283001 vvvv)
Absorption of the column effluent was monitored at 220 nm to detect the metabolites
Lower limit of quantification for R-95913 was 1 μM
Formation of R-138727 in human and dog intestinal and dog liver microsomes The
mixture (total volume 110 μL) in triplicate contained potassium phosphate buffer (43
mM pH 74) an NADPH generating system (13 mM NADP+ 33 mM
glucose-6-phosphate 04 unitsmL glucose-6-phosphate dehydrogenase and 33 mM
MgCl2) 5 mM glutathione and human or dog intestinal or dog liver microsomes (1 mg
proteinmL each) The mixture was preincubated at 37degC for 5 min and 11 μL of
R-95913 (final concentrations ranging from 16 to 200 μM) was added to the mixture
After incubation at 37degC for 15 min 200 μL of 5 mM methoxyphenacyl bromide
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solution and 100 μL of the IS solution (100 ngmL R-135766) in acetonitrile were added
to 100 μL of the reaction mixture to stop the reaction and the mixture was left for 10
min at room temperature to derivaitze a thiol moiety of R-138727 The mixture was
extracted by a solid phase extract column (Captiva Varian Inc Palo Alto CA) The
assay of R-138727 was performed following the methods previously reported (Hagihara
et al 2009) Separation of the analytes by HPLC was conducted using an
Alliance2690 Separations Module (Waters Co Milford MA) Mass spectra were
determined using a Quattro LC MSMS system (Micromass Ltd Milford MA) in the
positive ion detection mode using an ESI-interface A lower limit of quantification
was set at 16 nM Data acquisition and analyses were performed using MassLynx
software (Version 40 Micromass Ltd)
Data handling The formation pattern of R-138727 in human or dog intestinal or dog
liver microsomes appeared monophasic in each Eadie-Hofstee plot (data not shown)
Therefore the data were fitted to eq 1 using WinNonlin Professional (version 401
Pharsight Corp)
V = Vmax times S (Km + S) (6)
where S Km and Vmax is the substrate concentration Michaelis-Menten constant and
maximal formation rate respectively The intrinsic clearance (CLint) was calculated as
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a ratio of Vmax to Km These parameters are expressed as meanplusmnSD
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Results
Plasma concentrations and AUC0-2hs after intraduodenal administration of
prasugrel to cannulated dogs The plasma concentrations of R-95913 and R-138727
were determined by LC-MS after intraduodenal administration of prasugrel to the portal
and hepatic vein cannulated dogs at a dose of 1 mgkg Plasma concentrations of
prasugrel were not analyzed as prasugrel was not detected unmodified even in the portal
vein in a previous study (Hagihara et al 2009) The plasma concentration-time
profiles of R-95913 and R-138727 are shown in Figures 2 and 3 respectively The
AUC0-2h value of R-95913 in the portal vein (525plusmn234 nghmL) was much higher than
those in hepatic and systemic veins (32plusmn17 and 17plusmn8 nghmL respectively) The
AUC0-2h values of R-138727 in the portal hepatic and systemic veins were 564plusmn187
nghmL 529plusmn136 nghmL and 495plusmn233 nghmL respectively The bimodal peaks
were detected in the plasma concentration-time profiles of R-95913 (Figure 2) This
may indicate reabsorption of R-95913 from the intestine as in the case of rats which
showed enterohepatic circulation of 14C-prasugrel (Hagihara et al 2007)
Availability of R-95913 and R-138727 in the intestine and the liver The FaFg prasrarr
913 and Fh 913 values were 080plusmn023 and 008plusmn006 respectively (Table 1) The
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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9
Japan) The operating parameters of the MS detector were set as follows capillary
voltage 35 kV ion source temperature 120degC and desolvation temperature 350degC
The detection was performed in the multiple reaction monitoring (MRM) mode The
concentrations of each analyte in the samples were calculated using the computer
software MassLynx (Version 34 Micromass Ltd)
Pharmacokinetic analysis The pharmacokinetic parameters were calculated using the
computer program WinNonlin Professional (ver 401 Pharsight Corp Mountain View
CA) based on the non-compartmental method The area under the plasma
concentration-time curve up to 2 h (AUC0-2h) was calculated by the trapezoidal method
and are expressed as the meanplusmnSD
Calculation of availabililty of R-95913 The availability of R-95913 was calculated
using the following equations
Fa Fg prasrarr913 = Qp Rb (AUCpor913 - AUCsys913) Dose (1)
Fh 913 = AUChep913 AUCpor913 (2)
where Fa Fg prasrarr913 and Fh 913 are fraction of intestinal absorption of prasugrel fraction
of prasugrel hydrolysis to R-95913 in the intestine and availability of R-95913 in the
liver respectively AUCpor913 AUChep913 and AUCsys913 indicate the AUC0-2h values
of R-95913 in the portal hepatic and systemic veins respectively Qp is portal blood
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flow and Rb is the bloodplasma concentration ratio Fa Qp and Rb are set as 0968
(Hagihara et al 2007) 521 mLmin (Hoshino et al 1986) and 0597 (In-house data
Daiichi Sankyo Co Ltd) respectively
Calculation of availabililty of R-138727 The availability of R-138727 was calculated
using the following equations
Fa Fg prasrarr727 = Qp Rb (AUCpor727 - AUCsys727) Dose (3)
Fm 913rarr727 = (AUChep727 - AUCpor727 Fh 727 ) AUCpor913 (4)
Fa Fg prasrarr727 Fh 727 + Fa Fg prasrarr913 Fm 913rarr727 = Relative BA (5)
where Fg prasrarr727 Fm 913rarr727 and Fh 727 are fraction of prasugrel conversion to R-138727
in the intestine fraction of R-95913 conversion to R-138727 in the liver and availability
of R-138727 in the liver respectively AUCpor727 AUChep727 and AUCsys727 indicate
the AUC0-2h values of R-138727 in the portal hepatic and systemic veins respectively
Relative BA means the relative bioavailability of R-138727 after oral dosing of
prasugrel to dogs and was set as 248 (Hagihara et al 2009)
Formation of R-95913 in dog and human intestinal S9 The mixture (total volume
2475 μL) in triplicate contained potassium phosphate buffer (7 mM pH 74) an
NADPH generating system containing 25 mM β-NADP 25 mM G-6-P 05 unitsmL
G-6-PDH and 10 mM MgCl2 5 mM glutathione and dog or human intestinal S9 (10 mg
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proteinmL each) The mixture was preincubated at 37degC for 5 min and 25 μL of
prasugrel (final concentration 250 μM) was added to the mixture which was incubated
at 37degC for 0 5 15 30 and 60 min Ninety μL of ethanol was added to 30 μL of the
reaction mixture to stop the reaction at each time point and the mixture was centrifuged
(15000g 3 min 4degC) Twenty five μL of the supernatant was injected into the HPLC
system HPLC was performed using YMC-ODS A-302 (46 mm id times150 mm)
pumped at flow rate of 05 mLmin with a mobile phase consisting of acetonitrile
isopropyl alcohol distilled water trifluoroacetic acid (51283001 vvvv)
Absorption of the column effluent was monitored at 220 nm to detect the metabolites
Lower limit of quantification for R-95913 was 1 μM
Formation of R-138727 in human and dog intestinal and dog liver microsomes The
mixture (total volume 110 μL) in triplicate contained potassium phosphate buffer (43
mM pH 74) an NADPH generating system (13 mM NADP+ 33 mM
glucose-6-phosphate 04 unitsmL glucose-6-phosphate dehydrogenase and 33 mM
MgCl2) 5 mM glutathione and human or dog intestinal or dog liver microsomes (1 mg
proteinmL each) The mixture was preincubated at 37degC for 5 min and 11 μL of
R-95913 (final concentrations ranging from 16 to 200 μM) was added to the mixture
After incubation at 37degC for 15 min 200 μL of 5 mM methoxyphenacyl bromide
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solution and 100 μL of the IS solution (100 ngmL R-135766) in acetonitrile were added
to 100 μL of the reaction mixture to stop the reaction and the mixture was left for 10
min at room temperature to derivaitze a thiol moiety of R-138727 The mixture was
extracted by a solid phase extract column (Captiva Varian Inc Palo Alto CA) The
assay of R-138727 was performed following the methods previously reported (Hagihara
et al 2009) Separation of the analytes by HPLC was conducted using an
Alliance2690 Separations Module (Waters Co Milford MA) Mass spectra were
determined using a Quattro LC MSMS system (Micromass Ltd Milford MA) in the
positive ion detection mode using an ESI-interface A lower limit of quantification
was set at 16 nM Data acquisition and analyses were performed using MassLynx
software (Version 40 Micromass Ltd)
Data handling The formation pattern of R-138727 in human or dog intestinal or dog
liver microsomes appeared monophasic in each Eadie-Hofstee plot (data not shown)
Therefore the data were fitted to eq 1 using WinNonlin Professional (version 401
Pharsight Corp)
V = Vmax times S (Km + S) (6)
where S Km and Vmax is the substrate concentration Michaelis-Menten constant and
maximal formation rate respectively The intrinsic clearance (CLint) was calculated as
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a ratio of Vmax to Km These parameters are expressed as meanplusmnSD
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Results
Plasma concentrations and AUC0-2hs after intraduodenal administration of
prasugrel to cannulated dogs The plasma concentrations of R-95913 and R-138727
were determined by LC-MS after intraduodenal administration of prasugrel to the portal
and hepatic vein cannulated dogs at a dose of 1 mgkg Plasma concentrations of
prasugrel were not analyzed as prasugrel was not detected unmodified even in the portal
vein in a previous study (Hagihara et al 2009) The plasma concentration-time
profiles of R-95913 and R-138727 are shown in Figures 2 and 3 respectively The
AUC0-2h value of R-95913 in the portal vein (525plusmn234 nghmL) was much higher than
those in hepatic and systemic veins (32plusmn17 and 17plusmn8 nghmL respectively) The
AUC0-2h values of R-138727 in the portal hepatic and systemic veins were 564plusmn187
nghmL 529plusmn136 nghmL and 495plusmn233 nghmL respectively The bimodal peaks
were detected in the plasma concentration-time profiles of R-95913 (Figure 2) This
may indicate reabsorption of R-95913 from the intestine as in the case of rats which
showed enterohepatic circulation of 14C-prasugrel (Hagihara et al 2007)
Availability of R-95913 and R-138727 in the intestine and the liver The FaFg prasrarr
913 and Fh 913 values were 080plusmn023 and 008plusmn006 respectively (Table 1) The
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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10
flow and Rb is the bloodplasma concentration ratio Fa Qp and Rb are set as 0968
(Hagihara et al 2007) 521 mLmin (Hoshino et al 1986) and 0597 (In-house data
Daiichi Sankyo Co Ltd) respectively
Calculation of availabililty of R-138727 The availability of R-138727 was calculated
using the following equations
Fa Fg prasrarr727 = Qp Rb (AUCpor727 - AUCsys727) Dose (3)
Fm 913rarr727 = (AUChep727 - AUCpor727 Fh 727 ) AUCpor913 (4)
Fa Fg prasrarr727 Fh 727 + Fa Fg prasrarr913 Fm 913rarr727 = Relative BA (5)
where Fg prasrarr727 Fm 913rarr727 and Fh 727 are fraction of prasugrel conversion to R-138727
in the intestine fraction of R-95913 conversion to R-138727 in the liver and availability
of R-138727 in the liver respectively AUCpor727 AUChep727 and AUCsys727 indicate
the AUC0-2h values of R-138727 in the portal hepatic and systemic veins respectively
Relative BA means the relative bioavailability of R-138727 after oral dosing of
prasugrel to dogs and was set as 248 (Hagihara et al 2009)
Formation of R-95913 in dog and human intestinal S9 The mixture (total volume
2475 μL) in triplicate contained potassium phosphate buffer (7 mM pH 74) an
NADPH generating system containing 25 mM β-NADP 25 mM G-6-P 05 unitsmL
G-6-PDH and 10 mM MgCl2 5 mM glutathione and dog or human intestinal S9 (10 mg
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proteinmL each) The mixture was preincubated at 37degC for 5 min and 25 μL of
prasugrel (final concentration 250 μM) was added to the mixture which was incubated
at 37degC for 0 5 15 30 and 60 min Ninety μL of ethanol was added to 30 μL of the
reaction mixture to stop the reaction at each time point and the mixture was centrifuged
(15000g 3 min 4degC) Twenty five μL of the supernatant was injected into the HPLC
system HPLC was performed using YMC-ODS A-302 (46 mm id times150 mm)
pumped at flow rate of 05 mLmin with a mobile phase consisting of acetonitrile
isopropyl alcohol distilled water trifluoroacetic acid (51283001 vvvv)
Absorption of the column effluent was monitored at 220 nm to detect the metabolites
Lower limit of quantification for R-95913 was 1 μM
Formation of R-138727 in human and dog intestinal and dog liver microsomes The
mixture (total volume 110 μL) in triplicate contained potassium phosphate buffer (43
mM pH 74) an NADPH generating system (13 mM NADP+ 33 mM
glucose-6-phosphate 04 unitsmL glucose-6-phosphate dehydrogenase and 33 mM
MgCl2) 5 mM glutathione and human or dog intestinal or dog liver microsomes (1 mg
proteinmL each) The mixture was preincubated at 37degC for 5 min and 11 μL of
R-95913 (final concentrations ranging from 16 to 200 μM) was added to the mixture
After incubation at 37degC for 15 min 200 μL of 5 mM methoxyphenacyl bromide
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solution and 100 μL of the IS solution (100 ngmL R-135766) in acetonitrile were added
to 100 μL of the reaction mixture to stop the reaction and the mixture was left for 10
min at room temperature to derivaitze a thiol moiety of R-138727 The mixture was
extracted by a solid phase extract column (Captiva Varian Inc Palo Alto CA) The
assay of R-138727 was performed following the methods previously reported (Hagihara
et al 2009) Separation of the analytes by HPLC was conducted using an
Alliance2690 Separations Module (Waters Co Milford MA) Mass spectra were
determined using a Quattro LC MSMS system (Micromass Ltd Milford MA) in the
positive ion detection mode using an ESI-interface A lower limit of quantification
was set at 16 nM Data acquisition and analyses were performed using MassLynx
software (Version 40 Micromass Ltd)
Data handling The formation pattern of R-138727 in human or dog intestinal or dog
liver microsomes appeared monophasic in each Eadie-Hofstee plot (data not shown)
Therefore the data were fitted to eq 1 using WinNonlin Professional (version 401
Pharsight Corp)
V = Vmax times S (Km + S) (6)
where S Km and Vmax is the substrate concentration Michaelis-Menten constant and
maximal formation rate respectively The intrinsic clearance (CLint) was calculated as
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a ratio of Vmax to Km These parameters are expressed as meanplusmnSD
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Results
Plasma concentrations and AUC0-2hs after intraduodenal administration of
prasugrel to cannulated dogs The plasma concentrations of R-95913 and R-138727
were determined by LC-MS after intraduodenal administration of prasugrel to the portal
and hepatic vein cannulated dogs at a dose of 1 mgkg Plasma concentrations of
prasugrel were not analyzed as prasugrel was not detected unmodified even in the portal
vein in a previous study (Hagihara et al 2009) The plasma concentration-time
profiles of R-95913 and R-138727 are shown in Figures 2 and 3 respectively The
AUC0-2h value of R-95913 in the portal vein (525plusmn234 nghmL) was much higher than
those in hepatic and systemic veins (32plusmn17 and 17plusmn8 nghmL respectively) The
AUC0-2h values of R-138727 in the portal hepatic and systemic veins were 564plusmn187
nghmL 529plusmn136 nghmL and 495plusmn233 nghmL respectively The bimodal peaks
were detected in the plasma concentration-time profiles of R-95913 (Figure 2) This
may indicate reabsorption of R-95913 from the intestine as in the case of rats which
showed enterohepatic circulation of 14C-prasugrel (Hagihara et al 2007)
Availability of R-95913 and R-138727 in the intestine and the liver The FaFg prasrarr
913 and Fh 913 values were 080plusmn023 and 008plusmn006 respectively (Table 1) The
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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proteinmL each) The mixture was preincubated at 37degC for 5 min and 25 μL of
prasugrel (final concentration 250 μM) was added to the mixture which was incubated
at 37degC for 0 5 15 30 and 60 min Ninety μL of ethanol was added to 30 μL of the
reaction mixture to stop the reaction at each time point and the mixture was centrifuged
(15000g 3 min 4degC) Twenty five μL of the supernatant was injected into the HPLC
system HPLC was performed using YMC-ODS A-302 (46 mm id times150 mm)
pumped at flow rate of 05 mLmin with a mobile phase consisting of acetonitrile
isopropyl alcohol distilled water trifluoroacetic acid (51283001 vvvv)
Absorption of the column effluent was monitored at 220 nm to detect the metabolites
Lower limit of quantification for R-95913 was 1 μM
Formation of R-138727 in human and dog intestinal and dog liver microsomes The
mixture (total volume 110 μL) in triplicate contained potassium phosphate buffer (43
mM pH 74) an NADPH generating system (13 mM NADP+ 33 mM
glucose-6-phosphate 04 unitsmL glucose-6-phosphate dehydrogenase and 33 mM
MgCl2) 5 mM glutathione and human or dog intestinal or dog liver microsomes (1 mg
proteinmL each) The mixture was preincubated at 37degC for 5 min and 11 μL of
R-95913 (final concentrations ranging from 16 to 200 μM) was added to the mixture
After incubation at 37degC for 15 min 200 μL of 5 mM methoxyphenacyl bromide
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solution and 100 μL of the IS solution (100 ngmL R-135766) in acetonitrile were added
to 100 μL of the reaction mixture to stop the reaction and the mixture was left for 10
min at room temperature to derivaitze a thiol moiety of R-138727 The mixture was
extracted by a solid phase extract column (Captiva Varian Inc Palo Alto CA) The
assay of R-138727 was performed following the methods previously reported (Hagihara
et al 2009) Separation of the analytes by HPLC was conducted using an
Alliance2690 Separations Module (Waters Co Milford MA) Mass spectra were
determined using a Quattro LC MSMS system (Micromass Ltd Milford MA) in the
positive ion detection mode using an ESI-interface A lower limit of quantification
was set at 16 nM Data acquisition and analyses were performed using MassLynx
software (Version 40 Micromass Ltd)
Data handling The formation pattern of R-138727 in human or dog intestinal or dog
liver microsomes appeared monophasic in each Eadie-Hofstee plot (data not shown)
Therefore the data were fitted to eq 1 using WinNonlin Professional (version 401
Pharsight Corp)
V = Vmax times S (Km + S) (6)
where S Km and Vmax is the substrate concentration Michaelis-Menten constant and
maximal formation rate respectively The intrinsic clearance (CLint) was calculated as
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a ratio of Vmax to Km These parameters are expressed as meanplusmnSD
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Results
Plasma concentrations and AUC0-2hs after intraduodenal administration of
prasugrel to cannulated dogs The plasma concentrations of R-95913 and R-138727
were determined by LC-MS after intraduodenal administration of prasugrel to the portal
and hepatic vein cannulated dogs at a dose of 1 mgkg Plasma concentrations of
prasugrel were not analyzed as prasugrel was not detected unmodified even in the portal
vein in a previous study (Hagihara et al 2009) The plasma concentration-time
profiles of R-95913 and R-138727 are shown in Figures 2 and 3 respectively The
AUC0-2h value of R-95913 in the portal vein (525plusmn234 nghmL) was much higher than
those in hepatic and systemic veins (32plusmn17 and 17plusmn8 nghmL respectively) The
AUC0-2h values of R-138727 in the portal hepatic and systemic veins were 564plusmn187
nghmL 529plusmn136 nghmL and 495plusmn233 nghmL respectively The bimodal peaks
were detected in the plasma concentration-time profiles of R-95913 (Figure 2) This
may indicate reabsorption of R-95913 from the intestine as in the case of rats which
showed enterohepatic circulation of 14C-prasugrel (Hagihara et al 2007)
Availability of R-95913 and R-138727 in the intestine and the liver The FaFg prasrarr
913 and Fh 913 values were 080plusmn023 and 008plusmn006 respectively (Table 1) The
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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12
solution and 100 μL of the IS solution (100 ngmL R-135766) in acetonitrile were added
to 100 μL of the reaction mixture to stop the reaction and the mixture was left for 10
min at room temperature to derivaitze a thiol moiety of R-138727 The mixture was
extracted by a solid phase extract column (Captiva Varian Inc Palo Alto CA) The
assay of R-138727 was performed following the methods previously reported (Hagihara
et al 2009) Separation of the analytes by HPLC was conducted using an
Alliance2690 Separations Module (Waters Co Milford MA) Mass spectra were
determined using a Quattro LC MSMS system (Micromass Ltd Milford MA) in the
positive ion detection mode using an ESI-interface A lower limit of quantification
was set at 16 nM Data acquisition and analyses were performed using MassLynx
software (Version 40 Micromass Ltd)
Data handling The formation pattern of R-138727 in human or dog intestinal or dog
liver microsomes appeared monophasic in each Eadie-Hofstee plot (data not shown)
Therefore the data were fitted to eq 1 using WinNonlin Professional (version 401
Pharsight Corp)
V = Vmax times S (Km + S) (6)
where S Km and Vmax is the substrate concentration Michaelis-Menten constant and
maximal formation rate respectively The intrinsic clearance (CLint) was calculated as
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a ratio of Vmax to Km These parameters are expressed as meanplusmnSD
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Results
Plasma concentrations and AUC0-2hs after intraduodenal administration of
prasugrel to cannulated dogs The plasma concentrations of R-95913 and R-138727
were determined by LC-MS after intraduodenal administration of prasugrel to the portal
and hepatic vein cannulated dogs at a dose of 1 mgkg Plasma concentrations of
prasugrel were not analyzed as prasugrel was not detected unmodified even in the portal
vein in a previous study (Hagihara et al 2009) The plasma concentration-time
profiles of R-95913 and R-138727 are shown in Figures 2 and 3 respectively The
AUC0-2h value of R-95913 in the portal vein (525plusmn234 nghmL) was much higher than
those in hepatic and systemic veins (32plusmn17 and 17plusmn8 nghmL respectively) The
AUC0-2h values of R-138727 in the portal hepatic and systemic veins were 564plusmn187
nghmL 529plusmn136 nghmL and 495plusmn233 nghmL respectively The bimodal peaks
were detected in the plasma concentration-time profiles of R-95913 (Figure 2) This
may indicate reabsorption of R-95913 from the intestine as in the case of rats which
showed enterohepatic circulation of 14C-prasugrel (Hagihara et al 2007)
Availability of R-95913 and R-138727 in the intestine and the liver The FaFg prasrarr
913 and Fh 913 values were 080plusmn023 and 008plusmn006 respectively (Table 1) The
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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a ratio of Vmax to Km These parameters are expressed as meanplusmnSD
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Results
Plasma concentrations and AUC0-2hs after intraduodenal administration of
prasugrel to cannulated dogs The plasma concentrations of R-95913 and R-138727
were determined by LC-MS after intraduodenal administration of prasugrel to the portal
and hepatic vein cannulated dogs at a dose of 1 mgkg Plasma concentrations of
prasugrel were not analyzed as prasugrel was not detected unmodified even in the portal
vein in a previous study (Hagihara et al 2009) The plasma concentration-time
profiles of R-95913 and R-138727 are shown in Figures 2 and 3 respectively The
AUC0-2h value of R-95913 in the portal vein (525plusmn234 nghmL) was much higher than
those in hepatic and systemic veins (32plusmn17 and 17plusmn8 nghmL respectively) The
AUC0-2h values of R-138727 in the portal hepatic and systemic veins were 564plusmn187
nghmL 529plusmn136 nghmL and 495plusmn233 nghmL respectively The bimodal peaks
were detected in the plasma concentration-time profiles of R-95913 (Figure 2) This
may indicate reabsorption of R-95913 from the intestine as in the case of rats which
showed enterohepatic circulation of 14C-prasugrel (Hagihara et al 2007)
Availability of R-95913 and R-138727 in the intestine and the liver The FaFg prasrarr
913 and Fh 913 values were 080plusmn023 and 008plusmn006 respectively (Table 1) The
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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Results
Plasma concentrations and AUC0-2hs after intraduodenal administration of
prasugrel to cannulated dogs The plasma concentrations of R-95913 and R-138727
were determined by LC-MS after intraduodenal administration of prasugrel to the portal
and hepatic vein cannulated dogs at a dose of 1 mgkg Plasma concentrations of
prasugrel were not analyzed as prasugrel was not detected unmodified even in the portal
vein in a previous study (Hagihara et al 2009) The plasma concentration-time
profiles of R-95913 and R-138727 are shown in Figures 2 and 3 respectively The
AUC0-2h value of R-95913 in the portal vein (525plusmn234 nghmL) was much higher than
those in hepatic and systemic veins (32plusmn17 and 17plusmn8 nghmL respectively) The
AUC0-2h values of R-138727 in the portal hepatic and systemic veins were 564plusmn187
nghmL 529plusmn136 nghmL and 495plusmn233 nghmL respectively The bimodal peaks
were detected in the plasma concentration-time profiles of R-95913 (Figure 2) This
may indicate reabsorption of R-95913 from the intestine as in the case of rats which
showed enterohepatic circulation of 14C-prasugrel (Hagihara et al 2007)
Availability of R-95913 and R-138727 in the intestine and the liver The FaFg prasrarr
913 and Fh 913 values were 080plusmn023 and 008plusmn006 respectively (Table 1) The
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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at ASPE
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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at ASPE
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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ownloaded from
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at ASPE
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ownloaded from
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at ASPE
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FaFg prasrarr727 Fm 913rarr727 and Fh 727 values of R-138727 were 013plusmn013 023plusmn006 and
075plusmn021 respectively (Table 2) The dosed prasugrel was absorbed and converted to
R-95913 by 93 (FaFg prasrarr727 + FaFg prasrarr913) and to R-138727 by 13 in the
intestine R-95913 which passed through the intestine was converted to R-138727 by
23 in the liver (Figure 4)
Formation of R-95913 in dog and human intestinal S9 Prasugrel was almost
completely hydrolyzed to R-95913 at 5 min in dog and human intestinal S9 (Figure 5)
The results indicated comparable hydrolytic activities of prasugrel by dog and human
intestinal enzymes
Formation of R-138727 in dog and human intestinal and liver microsomes The
rates of R-138727 formation over a range of R-95913 concentrations were determined
in dog intestinal and liver microsomes and human intestinal microsomes Each
concentration - R-138727 fromation curve was well fitted to equation 6 (Figure 6)
Dog intestinal and liver microsomes and human intestinal microsomes exhibited
apparent Km values of 427 185 and 804 μM respectively The corresponding CLint
values in these microsomes were 12 206 and 11 μLminmg respectively The CLint
ratios (liverintestine) were 172 and 87 in dogs and humans respectively (Table 3)
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
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23
Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
DMD35956
24
Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
25
Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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Discussion
There have been several reports indicating that active metabolite of prasugrel is formed
in the intestine of humans Farid et al (2007a) showed that concomitant
administration of a potent CYP3A45 inhibitor ketoconazole with prasugrel resulted in
delayed appearance of R-138727 in plasma Also AUC of R-95913 doubled and Cmax
increased by 71 to 93 while tmax or t12 of R-95913 did not change (Farid et al
2007a) Considering that the AUC and Cmax reflect bioavailability whereas t12
depends directly on hepatic clearance these differential pharmacokinetic effects are
indicative of R-95913 as a substrate for intestinal CYP3A in humans Small et al
reported a clinical observation in patients with chronic liver disease where moderate
hepatic impairment appeared to have no effect on exposure to prasugrels active
metabolite R-138727 and little or no effect on platelet aggregation relative to healthy
controls (Small et al 2009) Based on this information we considered the intestine as
an important contributor to R-138727 formation in vivo Generally it is difficult to
perform quantitative kinetic analyses of a prodrug and its metabolites in vivo which
require complex models to describe respective concentration profiles (Tsukamoto et al
2001) Therefore we evaluated each metabolitersquos availability using a simple
calculation method without any differential equations The results in the present study
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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demonstrated quantitatively important contribution rates of the intestine to R-138727
formation in dogs where 13 of dosed prasugrel was converted to R-138727 in the
intestine Since availability of R-138727 in the liver was 75 about 10 (13
75) of dosed prasugrel is thought to reach the circulation as R-138727 via
bioactivation in the intestine The relative bioavailability of R-138727 after oral
administration of prasugrel to dogs is 25 (Hagihara et al 2009) and therefore
approximately 40 (1025) of exposure to R-138727 in the circulation is likely
attributed to intestinal bioactivation
In this study dosed prasugrel was calculated to be substantially converted to R-95913
(by 93) during the absorption process through the intestine which is consistent with
the previous clinical and non-clinical observations In human plasma prasugrel was
not detected unmodified due to rapid hydrolysis by carboxylesterases (Farid et al
2007b Williams et al 2008) Also a Caco-2 cell study showed the complete
conversion of prasugrel to R-95913 during absorption (Williams et al 2008) In the
current study carboxylesterase inhibitors were not added during blood sampling
indicating the possibility of hydrolysis of prasugrel in the extracted blood samples
However prasugrel is rapidly hydrolyzed in dog intestinal S9 (Figure 5) and therefore
prasugrel administered to dogs is thought to be hydrolyzed in the intestine in vivo
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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ownloaded from
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at ASPE
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ownloaded from
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at ASPE
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ownloaded from
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at ASPE
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ugust 30 2020dm
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ownloaded from
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We used dogs to evaluate respective fractions of prasugrel bioactivation in the intestine
and liver Since the existence of CYP3A activities have been reported in the intestine
and liver in dogs (Komura et al 2002 Sahi et al 2002) it is considered reasonable to
use dogs for assessment of the active metabolite formation of prasugrel which is
mediated mainly by CYP3A Indeed prasugrel was metabolized to R-138727 in dog
intestinal and liver microsomes with comparable CLint values to those in human
intestinal and liver microsomes respectively (Table 3) The ratios of CLint
(liverintestine) were 172 and 87 in dogs and humans respectively and these were
relatively higher than that of the fraction of R-138727 formation in dogs (Fm 913rarr727Fg
prasrarr727 = 18) Such discrepancy between in vitro and in vivo might be explained by
the difference of physiological conditions in intestine and liver (ie more absolute
exposure to the substrate in the intestine during absorption) or possibly higher unbound
fraction of the substrate in the intestine In dog small intestine no carboxylesterases
were found in the previous report (Taketani et al 2007) However prasugrel was
hydrolyzed in dog intestinal S9 to the same degree as in human intestinal S9 (Figure 5)
indicating the existence of complementary esterases in dog intestine This concept is
also supported by the previous observation that prasugrel was not detected unmodified
in the portal vein after oral administration of prasugrel to dogs (Hagihara et al 2009)
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
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25
Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
DMD35956
26
Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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at ASPE
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ownloaded from
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27
Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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at ASPE
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ownloaded from
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at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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Thus a similar fraction of R-138727 formed in the intestine in dogs could be expected
in humans as well
Clopidogrel seems not to be converted to either thiolactone intermediate or active
metabolite in the intestine (Hagihara et al 2009 Kazui et al 2010) This could be the
reason for slower onset of inhibitory effect on platelet aggregation by clopidogrel than
prasugrel in the clinical studies (Payne et al 2007 Wallentin et al 2008)
In conclusion a significant portion of R-95913 was oxidized to R-138727 during
intestinal absorption in dogs This is the first report to quantitatively evaluate the
contribution of the intestine and liver to the formation of R-95913 and R-138727 and
the calculation method used in this study may provide a useful tool for evaluation of the
bioactivation of other prodrugs in the liver and intestine
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Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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at ASPE
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ownloaded from
DMD35956
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
29
FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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at ASPE
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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at ASPE
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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DMD35956
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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DMD35956
20
Acknowledgements
The authors thank Drs Takashi Ito and Daisuke Nakai of Daiichi Sankyo Co Ltd and
Drs Mary Pat Knadler and Steven A Wrighton of Eli Lilly and Company for their
helpful comments and discussion on this study
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
DMD35956
21
Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
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22
REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
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23
Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
25
Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
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26
Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
27
Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
28
FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
29
FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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at ASPE
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
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31
Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
32
Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
33
Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
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at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
daspetjournalsorgD
ownloaded from
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at ASPE
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ugust 30 2020dm
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Authorship Contributions
Participated in research design Hagihara Kazui Ikenaga Nanba Fusegawa Izumi
Ikeda and Kurihara
Conducted experiments Hagihara Kazui Ikenaga Nanba and Fusegawa
Performed data analysis Hagihara and Kazui
Wrote or contributed to the writing of the manuscript Hagihara Kazui Ikenaga Izumi
Ikeda and Kurihara
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at ASPE
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ugust 30 2020dm
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22
REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
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23
Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
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24
Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
25
Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
26
Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
27
Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
28
FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
29
FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
DMD35956
32
Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
33
Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
22
REFERENCES
Baker JAR Oluyedun OA Farid NA Ring BJ Wrighton SA Kurihara A Guo Y (2008)
Formation of the isomers of the active metabolite of prasugrel by allelic variants of the
human cytochrome P450 isozymes Drug Metab Rev 40 (suppl 3)332
Ernest CS 2nd Small DS Rohatagi S Salazar DE Wallentin L Winters KJ Wrishko
RE (2008) Population pharmacokinetics and pharmacodynamics of prasugrel and
clopidogrel in aspirin-treated patients with stable coronary artery disease J
Pharmacokinet Pharmacodyn 35593-618
Farid NA Payne CD Small DS Winters KJ Ernest CS 2nd Brandt JT Darstein C
Jakubowski JA Salazar DE (2007a) Cytochrome P4503A inhibition by ketoconazole
affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently
Clin Pharmacol Ther81735-741
Farid NA Smith RL Gillespie TA Rash TJ Blair PE Kurihara A Goldberg MJ
(2007b) The disposition of prasugrel a novel thienopyridine in humans Drug Metab
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
23
Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
24
Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
25
Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
26
Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
27
Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
28
FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
29
FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
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ownloaded from
DMD35956
30
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
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31
Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
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ownloaded from
DMD35956
32
Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
33
Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
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at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
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at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
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at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
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at ASPE
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Dispos 351096-1104
Farid NA Kurihara A Wrighton SA (2010) Metabolism and disposition of the
thienopyridine antiplatelet drugs ticlopidine clopidogrel and prasugrel in humans J
Clin Pharmacol 50126-142
Hagihara K Kurihara A Kawai K Kazui M Takahashi M Kawabata K Farid NA
Ikeda T (2007) Absorption distribution and excretion of the new thienopyridine agent
prasugrel in rats Xenobiotica 37788-801
Hagihara K Kazui M Ikenaga H Nanba T Fusegawa K Takahashi M Kurihara A
Okazaki O Farid NA Ikeda T (2009) Comparison of formation of thiolactones and
active metabolites of prasugrel and clopidogrel in rats and dogs Xenobiotica
39218-226
Hoshino S Nonami T Kato T Asahi K Nakao A Suenaga M Takagi H (1986)
Experimental study of portal blood flow measurement by local thermodilution method
-the influence of Dobutamine and Dapamine on portal hemodynamics- Jpn J
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at ASPE
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Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
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ownloaded from
DMD35956
25
Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
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26
Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
27
Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
28
FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
29
FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
DMD35956
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ownloaded from
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32
Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
DMD35956
33
Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
DMD35956
24
Gastroenterol Surg 19 914-919
Kazui M Kurihara A Hagihara K Farid NA Ikeda T (2005) Prasugrel (CS-747
LY640315) a novel thienopyridine antiplatelet agent more efficiently generates active
metabolite compared to clopidogrel Drug Metab Rev 37(suppl 2)98-99
Komura H Yasuda M Yoshida NH Sugiyama Y (2002) Species difference in
nisoldipine oxidation activity in the small intestine Drug Metab Pharmacokinet
17427-436
Paine MF Hart HL Ludington SS Haining RL Rettie AE Zeldin DC (2006) The
human intestinal cytochrome P450 ldquopierdquo Drug Metab Dispos 34880-886
Payne CD Li YG Small DS Ernest CS 2nd Farid NA Jakubowski JA Brandt JT
Salazar DE Winters KJ (2007) Increased active metabolite formation explains the
greater platelet inhibition with prasugrel compared to high-dose clopidogrel J
Cardiovasc Pharmacol 50555-562
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
25
Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
26
Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
27
Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
28
FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
29
FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
30
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
DMD35956
31
Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
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ownloaded from
DMD35956
32
Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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Rehmel JL Eckstein JA Farid NA Heim JB Kasper SC Kurihara A Wrighton SA
Ring BJ (2006) Interactions of two major metabolites of prasugrel a thienopyridine
antiplatelet agent with the cytochromes P450 Drug Metab Dispos 34 600-607
Sahi J Reyner EL Bauman JN Gueneva-Boucheva K Burleigh JE Thomas VH (2002)
The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs
Drug Metab Dispos 30135-140
Small DS Farid NA Li YG Ernest CS 2nd Winters KJ Salazar DE Payne CD (2009)
Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver
disease J Clin Pharm Ther 34575-583
Sugidachi A Ogawa T Kurihara A Hagihara K Jakubowski JA Hashimoto M Niitsu
Y Asai F (2007) The greater in vivo antiplatelet effects of prasugrel as compared to
clopidogrel reflect more efficient generation of its active metabolite with similar
antiplatelet activity to that of clopidogrels active metabolite J Thromb Haemost
51545-1451
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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at ASPE
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ownloaded from
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Taketani M Shii M Ohura K Ninomiya S Imai T (2007) Carboxylesterase in the liver
and small intestine of experimental animals and human Life Sci 81924-932
Tsukamoto Y Kato Y Ura M Horii I Ishitsuka H Kusuhara H Sugiyama Y (2001) A
physiologically based pharmacokinetic analysis of capecitabine a triple prodrug of
5-FU in humans the mechanism for tumor-selective accumulation of 5-FU Pharm Res
18 1190-1202
Wallentin L Varenhorst C James S Erlinge D Braun OO Jakubowski JA Sugidachi A
Winters KJ Siegbahn A (2008) Prasugrel achieves greater and faster
P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient
generation of its active metabolite in aspirin-treated patients with coronary artery
disease Eur Heart J 2921-30
Williams ET Jones KO Ponsler GD Lowery SM Perkins EJ Wrighton SA
Ruterbories KJ Kazui M Farid NA (2008) The biotransformation of prasugrel a new
thienopyridine prodrug by the human carboxylesterases 1 and 2 Drug Metab Dispos
361227-32
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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at ASPE
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Wiviott SD Braunwald E McCabe CH Montalescot G Ruzyllo W Gottlieb S
Neumann FJ Ardissino D De Servi S Murphy SA Riesmeyer J Weerakkody G
Gibson CM Antman EM TRITON-TIMI 38 Investigators (2007) Prasugrel versus
clopidogrel in patients with acute coronary syndromes N Engl J Med 3572001-2015
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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at ASPE
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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at ASPE
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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at ASPE
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FOOTNOTES
A portion of this work was presented at the scientific session of International Society on
Thrombosis and Haemostasis Boston MA USA in 2009
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at ASPE
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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at ASPE
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ownloaded from
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at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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FIGURE LEGENDS
Figure 1 Bioactivation pathway of prasugrel
Figure 2 Plasma concentrations of R-95913 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-95913 in the portal vein (closed triangle) hepatic vein (open circle)
and systemic vein (cross) were determined by LC-MSMS The data are expressed as
the mean+SD
Figure 3 Plasma concentrations of R-138727 after intraduodenal administration of
prasugrel to dogs at a dose of 1 mgkg
Concentrations of R-138727 in the portal vein (closed triangle) hepatic vein (open
circle) and systemic vein (cross) were determined by LC-MSMS The data are
expressed as the mean+SD
Figure 4 Availability of R-95913 and R-138727 in the intestine and the liver in dogs
Fa fraction of intestinal absorption of prasugrel
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
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Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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at ASPE
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at ASPE
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at ASPE
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at ASPE
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at ASPE
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Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh 913 availability of R-95913 in the liver
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh 727 availability of R-138727 in the liver
Figure 5 Formation of R-95913 from prasugrel in dog and human intestinal S9
Prasugrel (250 μM) was incubated in dog and human intestinal S9 and the
concentrations of R-95913 were determined
Figure 6 Kinetic analyses of the formation of R-138727 from R-95913 in dog and
human intestinal and dog liver microsomes
The formation rates of R-138727 from R-95913 in triplicate were determined in dog
intestinal (a) and liver (b) microsomes and human intestinal microsomes (c)
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
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33
Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
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at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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ugust 30 2020dm
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ownloaded from
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Table 1 Availability of R-95913 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
913 Fh913 Fg prasrarr
913
Mean 080 008 083SD 023 006 023
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr913 fraction of prasugrel hydrolysis to R-95913 in the intestine
Fh913 availability of R-95913 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
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32
Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
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33
Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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ugust 30 2020dm
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at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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ugust 30 2020dm
daspetjournalsorgD
ownloaded from
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at ASPE
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ugust 30 2020dm
daspetjournalsorgD
ownloaded from
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at ASPE
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Table 2 Availability of R-138727 in the intestine and the liver after intraduodenal
administration of prasugrel to dogs at a dose of 1 mgkg
Fa Fg prasrarr
727 Fg prasrarr
727 Fm 913rarr
727 Fh727
Mean 013 013 023 075SD 013 013 006 021
Fa fraction of intestinal absorption of prasugrel
Fg prasrarr727 fraction of prasugrel conversion to R-138727 in the intestine
Fm 913rarr727 fraction of R-95913 conversion to R-138727 in the liver
Fh727 availability of R-138727 in the liver
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
DMD35956
33
Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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ugust 30 2020dm
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at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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ugust 30 2020dm
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ownloaded from
DMD35956
33
Table 3 Kinetic parameters for the formation of R-138727 in dog and human intestinal
and liver microsomes
Values for Km and Vmax are reported as the mean of parameter estimatesplusmnSD
Incubation time Km Vmax CLint CLint ratiomin microM pmolminmg microLminmg (liverintestine)
Intestinal microsome 15 427 plusmn 10 498 plusmn 20 12 plusmn 00Liver microsome 15 185 plusmn 06 3799 plusmn 207 206 plusmn 05Intestinal microsome 15 804 plusmn 115 892 plusmn 13 11 plusmn 01Liver microsome 15 26 247 96
Rehmel et al (2006)
87
172
Human
Dog
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
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ugust 30 2020dm
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ownloaded from
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at ASPE
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at ASPE
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at ASPE
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T Journals on A
ugust 30 2020dm
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This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
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This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
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T Journals on A
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This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
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ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
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daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from
This article has not been copyedited and formatted The final version may differ from this versionDMD Fast Forward Published on December 28 2010 as DOI 101124dmd110035956
at ASPE
T Journals on A
ugust 30 2020dm
daspetjournalsorgD
ownloaded from